Compositions and methods for growth factor modulation

ABSTRACT

Provided herein are proteins, antibodies, assays and methods useful for modulating growth factor levels and/or activities. In some embodiments, such growth factors are members of the TGF-β superfamily of proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 14/795,033, now U.S. Pat. No. 9,399,656, filed Jul. 9, 2015entitled Compositions and Methods for Growth Factor Modulation, which isa continuation of International Application No. PCT/US2014/036933, filedMay 6, 2014 entitled Compositions and Methods for Growth FactorModulation, which claims priority to U.S. Provisional Patent ApplicationNo. 61/819,840 filed May 6, 2013, entitled Compositions and Methods forGrowth Factor Modulation, U.S. Provisional Patent Application No.61/823,552 filed May 15, 2013, entitled Compositions and Methods forGrowth Factor Modulation and U.S. Provisional Patent Application No.61/900,438 filed Nov. 6, 2013, entitled Compositions and Methods forGrowth Factor Modulation, the contents of each of which are hereinincorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 17, 2016, isnamed 2035_1001USDIV_SL.txt and is 705,464 bytes in size.

FIELD OF THE INVENTION

Embodiments of the present invention may include recombinant proteins aswell as antibodies directed to such proteins. In some embodiments, suchproteins and antibodies may be related to the field of TGF-β familymember biology.

BACKGROUND OF THE INVENTION

Cell signaling molecules stimulate a variety of cellular activities.Such signaling is often tightly regulated, often through interactionswith other biomolecules, the extracellular and/or cellular matrix orwithin a particular cell environment or niche. Such interactions may bedirect or indirect.

Cell signaling cascades are involved in a number of diverse biologicalpathways including, but not limited to modulation of cell growth,modulation of tissue homeostasis, extracellular matrix (ECM) dynamics,modulation of cell migration, invasion and immunemodulation/suppression. In some cases, proteins involved in cellsignaling are synthesized and/or are sequestered in latent form,requiring stimulus of some kind to participate in signaling events.There remains a need in the art for agents, tools and methods formodulating cell signaling and/or cellular activities.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides recombinant proteinscomprising one or more TGF-β-related proteins comprising one or moreprotein modules selected from the group consisting of growth factorprodomain complexes (GPCs), latency associated peptides (LAPs), LAP-likedomains, straight jacket regions, growth factor domains, fastenerregions, furin cleavage site regions, arm regions, fingers regions,N-terminal regions for extracellular associations, latency loops, alpha1 helical regions, alpha 2 helical regions, RGD sequence regions,trigger loop regions and bowtie regions. In some embodiments,recombinant proteins of the present invention may comprise one or moreprotein modules from a vertebrate species. In some embodiments,recombinant proteins of the present invention may comprise one or moreprotein modules comprising one or more mutations. In some embodiments,recombinant proteins of the present invention may comprise one or moremutations comprising one or more furin cleavage site regions. In someembodiments, such mutations may prevent enzymatic cleavage ofrecombinant proteins of the present invention. In some embodiments,recombinant proteins of the present invention may comprise one or moremutations comprising a mutation of the amino acid sequence RXXR to theamino acid sequence RXG. In some embodiments, recombinant proteins ofthe present invention may comprise one or more mutations comprising amutation of the amino acid sequence RXXR to the amino acid sequenceAXXA. In some embodiments, recombinant proteins of the present inventionmay comprise one or more mutations comprising N-terminal regions forextracellular associations. In some embodiments, recombinant proteins ofthe present invention may comprise one or more mutations comprisingsubstitution and/or deletion of at least one cysteine residue presentwithin about the first 4, 5, 6 or 7 N-terminal amino acid residues. Insome embodiments, recombinant proteins of the present invention maycomprise one or more substitution of at least one cysteine residue withat least one serine residue.

In some embodiments, recombinant proteins of the present invention maybe complexed with a protein selected from the group consisting of LTBP1,LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3,fibrillin-4, GARP, LRRC33 and a combination or fragment thereof. In someembodiments, recombinant proteins of the present invention may compriseone or more detectable labels. Such detectable labels may comprisebiotin labels, polyhistidine tags and/or flag tags.

In some embodiments, the present invention provides chimeric proteinscomprising one or more protein modules from at least two TGF-β-relatedproteins wherein said protein modules may be selected from the groupconsisting of growth factor prodomain complexes (GPCs), latencyassociated peptides (LAPs), LAP-like domains, straight jacket regions,growth factor domains, fastener regions, furin cleavage site regions,arm regions, fingers regions, N-terminal regions for extracellularassociations, latency loops, alpha 1 helical regions, RGD sequenceregions, trigger loop regions, bowtie regions and any of those listed inTables 2, 3 and 11. In some embodiments, chimeric proteins of thepresent invention may comprise one or more protein modules selected fromone or more vertebrate species. In some embodiments, chimeric proteinsof the present invention may comprise GPCs. In some embodiments, suchGPCs may comprise at least one LAP or LAP-like domain from a TGF-βfamily member and at least one growth factor domain from a TGF-β familymember wherein the LAP or LAP-like domain and the growth factor domainare from different TGF-β family members. In some embodiments, chimericproteins of the present invention may comprise at least one LAP orLAP-like domain and at least one growth factor domain, each of which isselected from the group consisting of TGF-β1, TGF-β2, TGF-β3, GDF-8,GDF-11 and inhibin beta A. In some embodiments, chimeric proteins of thepresent invention may comprise one or more GPC wherein at least oneN-terminal region is from a TGF-β family member, at least one C-terminalregion is from a TGF-β family member and wherein the N-terminal regionand C-terminal region are from different TGF-β family members. In someembodiments, chimeric proteins of the present invention may comprise atleast one N-terminal region and at least one C-terminal region selectedfrom TGF-β1 terminal regions, TGF-β2 terminal regions, TGF-β3 terminalregions, GDF-8 terminal regions, GDF-11 terminal regions and inhibinbeta A terminal regions. In some embodiments, chimeric proteins of thepresent invention may comprise a GPC from at least one TGF-β familymember comprising at least one arm region from a different TGF-β familymember. In some embodiments, chimeric proteins of the present inventionmay comprise a GPC comprising at least one TGF-β family membercomprising at least one trigger loop region from a different TGF-βfamily member. In some embodiments, chimeric protein of the presentinvention may comprise any of the protein module combinations listed inTable 12.

In some embodiments, chimeric protein of the present invention may becomplexed with a protein selected from the group consisting of LTBP1,LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3,fibrillin-4, GARP and LRRC33 and a combination or fragment thereof. Insome embodiments, chimeric proteins of the present invention maycomprise one or more detectable labels. In some embodiments, suchdetectable labels may comprise at least one biotin label, polyhistidinetag and/or flag tag.

In some embodiments, the present invention provides an antibody directedto any of the recombinant proteins and/or chimeric proteins disclosedherein. In some embodiments, such antibodies comprise monoclonalantibodies. In some embodiments, antibodies of the present invention aresubstantially isolated. In some embodiments, monoclonal antibodies ofthe present invention are stabilizing antibodies. In some embodiments,stabilizing antibodies of the present invention reduce the level of freegrowth factor relative to the level of growth factor associated with oneor more GPC. In some embodiments, stabilizing antibodies may reducegrowth factor-dependent cellular signaling. In some embodiments,monoclonal antibodies of the present invention may comprise releasingantibodies. Such antibodies may increase the level of free growth factorrelative to the level of growth factor associated with one or more GPC.In some embodiments, releasing antibodies of the present invention mayincrease growth factor-dependent cellular signaling.

In some embodiments, the present invention provides compositionscomprising one or more of any of the recombinant proteins, one or moreof any of the chimeric proteins and/or one or more of any of theantibodies described herein combined with at least one excipient.

In some embodiments, the present invention provides methods ofmodulating the level of free growth factor in a subject or cell nichecomprising the use of one or more compositions described herein. In somesuch methods, the level of growth factor signaling is modulated.

In some embodiments, the present invention provides methods forselecting a desired antibody comprising the use of one or more assays,wherein such assays comprise one or more recombinant protein of theinvention. Some such methods comprise the steps of 1) providing anantibody binding assay, 2) contacting the binding assay with one or morecandidate antibodies, 3) obtaining binding data related to candidateantibody affinity for the one or more recombinant protein and 4)selecting a desired antibody based on the binding data. Binding assaysaccording to such methods may include an enzyme-linked immunosorbentassay (ELISA) and/or a fluorescence-associated cell sorting (FACS)-basedassay. In some cases, recombinant proteins of such assays may becomplexed with a protein selected from the group consisting of SEQ IDNOs: 153-161 and 286-292 or complexed with a protein selected from thegroup consisting of LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4, fibrillin-1,fibrillin-2, fibrillin-3, fibrillin-4, GARP, LRRC33, perlecan, decorin,elastin and collagen. In some cases, recombinant proteins may comprise achimeric protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 199-236 and 273.

Other methods of selecting a desired antibody may comprise the stepsof 1) providing a growth factor activity assay, 2) contacting the growthfactor activity assay with one or more candidate antibodies, 3)obtaining growth factor activity data and 4) selecting a desiredantibody based on the growth factor activity data. Growth factoractivity assays according to such methods may comprise cell-based assaysselected from the group consisting of luciferase-based assays andproliferation assays. Such cell-based assays may comprise one or moreexpression cells that express one or more recombinant protein of theinvention or a complex thereof. Such assays may further comprise one ormore responsive cells that yield gene expression data and/or viabilitydata.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising one or more of any of the recombinant proteinsdescribed herein, one or more of any of the chimeric proteins describedherein and/or one or more of any of the antibodies described herein andat least one pharmaceutically excipient.

Some methods of the invention comprise treatment of a TGF-β-relatedindication in a subject comprising contacting said subject with acomposition of the invention. TGF-β-related indications may includefibrotic indications (e.g. lung fibrosis, kidney fibrosis, liverfibrosis, cardiovascular fibrosis, skin fibrosis, and bone marrowfibrosis), myelofibrosis, cancer or cancer-related conditions (e.g.colon cancer, renal cancer, breast cancer, malignant melanoma andglioblastoma) and muscle disorders and/or injuries [e.g. cachexia,muscular dystrophy, chronic obstructive pulmonary disease (COPD), motorneuron disease, trauma, neurodegenerative disease, infection, rheumatoidarthritis, immobilization, sarcopenia, inclusion body myositis anddiabetes.]

In some embodiments, the invention provides a kit comprising acomposition of the invention and instructions for use thereof.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of various embodiments of the invention.

FIG. 1 is a diagram of the TGF-beta superfamily tree, where divergenceis proportional to branch length.

FIG. 2 is a schematic of one embodiment of a linear representation of atranslated growth factor monomer. In such embodiments, translated growthfactors may comprise secretion signal peptides, prodomains and growthfactor domains. In embodiments according to embodiment depicted here,translated growth factors may also comprise a cleavage site betweenprodomain and growth factor regions.

FIG. 3 is a schematic of one embodiment of a growth factor-prodomaincomplex (GPC) as well as an embodiment of a free growth factor dimer anda free latency associated peptide (LAP) dimer. The arrow indicates theability of proteins according to this embodiment to alter between freeand complexed forms.

FIG. 4 is a schematic of one embodiment of a free LAP dimer and a freegrowth factor dimer with labeled features and/or protein modules.

FIG. 5 is a schematic of an embodiment of a recombinant GPC.

FIG. 6 is a schematic of embodiments of mutant recombinant GPCs.

FIG. 7 depicts schematic representations of five recombinant proteinsalone or in complex with LTBP or GARP.

FIGS. 8A-8G show structure-based alignment between TGF-β family memberproteins (SEQ ID NOS 1, 117, 116, 296, 2-4, 137, 5, 131, 125, 6, 14, 21,23-24, 27, 26, 28, and 10, respectively, in order of appearance)[adapted from Shi et al (Shi, M. et al., Latent TGF-β structure andactivation. Nature. 2011 Jun. 15; 474(7351):343-9, the contents of whichare herein incorporated by reference in their entirety.)] Cysteineresidues required for interaction with LTBPs and/or GARPs are boxed.Residues mutated in Camurati-Engelmann syndrome are indicated with astar. Protease cleavage sites are indicated with an up arrow. Proteinmodules and secondary structural elements are indicated with solid bars.Residues underlined at the N-terminus of GDF-8 correspond toalternatively predicted signal peptide processing sites. “Chimericmodule breakpoints” indicate regions where structural features areconserved and provide modules for chimeric protein construction(swapping of modules between family members) in all family members.N-terminal regions are shown in FIGS. 8A and 8B, internal regions areshown in FIGS. 8C and 8D and C-terminal regions are shown in FIGS.8E-8G.

FIGS. 9A-9C present 3 tables showing the percent identity between aminoacid sequences found in the TGF-β family. FIG. 9A demonstrates percentidentity among pro-proteins (prodomain and growth factor.) Percentidentity among growth factor domains is presented in FIG. 9B whilepercent identity among prodomains is presented in FIG. 9C.

FIG. 10 presents an alignment conducted between GDF-8 (myostatin) (SEQID NO: 5), GDF-11 (SEQ ID NO: 4), Inhibin A (SEQ ID NO: 6) and a GDF-8dimer (SEQ ID NO: 297). Arrows indicate cleavage sites. Regions involvedin internal interactions are boxed. Solid rectangles appear aboveresidues predicted to be involved in steric clashes in chimericconstructs. Stars denote important break points in protein modules.

FIG. 11 depicts the expression and purification of recombinant antigensand antigen complexes (Coomassie Blue stained SDS-PAGE).

FIG. 12 presents results from analyses of cell lines stably expressingTGF-β1/GARP complexes. 300.19 cells stably transfected with empty vectorcontrol (A), proTGF-β1-GARP (B) or TGF-β1 LAP-GARP (C) werefluorescently labeled with antibodies directed to expressed proteins andexamined for fluorescence intensity by flow cytometry. Luciferase assaydata is presented in (D) showing TGF-β signaling activity resulting fromco-culture of these cells with cells expressing αvβ₆ integrin.

FIG. 13 depicts recombinant histidine-tagged proGDF-8, separated bySDS-PAGE under reducing and non-reducing conditions, as visualized byCoomassie staining.

DETAILED DESCRIPTION

Growth factors are cell signaling molecules that stimulate a variety ofcellular activities. Due to their broad-reaching influence withinbiological systems, growth factor signaling is tightly regulated, oftenthrough interactions with other biomolecules, the extracellular and/orcellular matrix or within a particular cell environment or niche. Theseinteractions may be direct or indirect.

Growth factors of the transforming growth factor beta (TGF-β) family areinvolved in a variety of cellular processes. Growth factor binding totype II receptors leads to type I receptor phosphorylation andactivation (Denicourt, C. et al., Another twist in the transforminggrowth factor β-induced cell-cycle arrest chronicle. PNAS. 2003.100(26):15290-1.) Activated type I receptors may in turn phosphorylatereceptor-associated SMADs (R-SMADs) promoting co-SMAD (e.g. SMAD4)dimer/trimer formation and nuclear translocation. SMAD complexescollaborate with cofactors to modulate expression of TGF-β family membertarget genes.

TGF-β family member signaling cascades are involved in a number ofdiverse biological pathways including, but not limited to inhibition ofcell growth, tissue homeostasis, extracellular matrix (ECM) remodeling,endothelial to mesenchymal transition (EMT) in cell migration andinvasion and immune modulation/suppression as well as in mesenchymal toepithelial transition. TGF-β signaling related to growth inhibition andtissue homeostasis may affect epithelial, endothelial, hematopoietic andimmune cells through the activation of p21 and p15^(INK) to mediate cellcycle arrest and repress myc. In relation to ECM remodeling, TGF-βsignaling may increase fibroblast populations and ECM deposition (e.g.collagen). TGF-β signaling related to cell migration and invasion mayaffect epithelial and/or endothelial cells, inducing stem cell-likephenotypes. This aspect of signaling may play a role in smooth musclecell proliferation following vascular surgery and/or stenting. In theimmune system, TGF-β ligand is necessary for T regulatory cell functionand maintenance of immune precursor cell growth and homeostasis. Nearlyall immune cells comprise receptors for TGF-β and TGF-β knockout micedie postnataly due in part to inflammatory pathologies. Finally, TGF-βsuppresses interferon gamma-induced activation of natural killer cells(Wi, J. et al., 2011. Hepatology. 53(4):1342-51, the contents of whichare herein incorporated by reference in their entirety.)

The recent solution of the crystal structure of the latent form ofTGF-beta is a first for the entire TGF-beta family and offers deepinsights into these complexes (Shi, M. et al., Latent TGF-β structureand activation. Nature. 2011 Jun. 15; 474(7351):343-9). Almost allsignaling in the TGF-beta family goes through a common pathway whereby adimeric ligand is recognized by a heterotetrameric receptor complexcontaining two type I and two type II receptors. Each receptor has aserine-threonine kinase domain. Type II receptors phosphorylate type Ireceptors, which in turn phosphorylate receptor-regulated Smads thattranslocate to and accumulate in the nucleus and regulate transcription.

There are 33 different members of the TGF-beta family in humans (FIG.1). Members include the bone morphogenetic proteins (BMP), inhibin,activin, growth and differentiation factor (GDF), myostatin, nodal,anti-Mullerian hormone, and lefty proteins. A review of TGF-β familymembers, related signaling molecules as well as their relationships canbe found in Massague., 2000. Nature Reviews Molecular Cell Biology.1:169-78, the contents of which are herein incorporated by reference intheir entirety. In some embodiments, mature growth factors aresynthesized along with their prodomains as single polypeptide chains(see FIG. 2). In some embodiments, such polypeptide chains may comprisecleavage sites for separation of prodomains from mature growth factors.In some embodiments, such cleavage sites are furin cleavage sitesrecognized and cleaved by proprotein convertases.

In general, homology among TGF-β family member growth factor domains isrelatively high. Interestingly, prodomain homology is much lower. Thislack of homology may be an important factor in altered growth factorregulation among family members. In some cases, prodomains may guideproper folding and/or dimerization of growth factor domains. Prodomainshave very recently been recognized, in some cases, to have importantfunctions in directing growth factors (after secretion) to specificlocations in the extracellular matrix (ECM) and/or cellular matrix,until other signals are received that cause growth factor release fromlatency. Release from latency may occur in highly localized environmentswhereby growth factors may act over short distances (e.g. from about 1cell diameter to about a few cell diameters, from about 2 cell diametersto about 100 cell diameters and/or from about 10 cell diameters to about10,000 cell diameters) and cleared once they reach the circulation. Somegrowth factor-prodomain complexes are secreted as homodimers. In someembodiments, prodomain-growth factor complexes may be secreted asheterodimers.

As used herein, the term “TGF-β-related protein” refers to a TGF-βisoform, a TGF-β family member or a TGF-β family member-related protein.TGF-β family members may include, but are not limited to any of thoseshown in FIG. 1 and/or listed in Table 1. These include, but are notlimited to TGF-β proteins, BMPs, myostatin, GDFs and inhibins. In someembodiments, the present invention provides tools and/or methods forisolating, characterizing and or modulating TGF-β-related proteins.Aspects of the present invention provide tools and/or methods forcharacterizing and/or modulating cellular activities related toTGF-β-related protein signaling. In other embodiments, tools of thepresent invention may comprise antigens comprising one or morecomponents of one or more TGF-β-related proteins. Some tools maycomprise antibodies directed toward antigens of the present invention.In additional embodiments, tools of the present invention may compriseassays for the detection and/or characterization of TGF-β-relatedproteins, the detection and/or characterization of antibodies directedtoward TGF-β-related proteins and/or the detection and/orcharacterization of cellular activities and/or their cellular signalingrelated to TGF-β-related proteins.

Proteins of Interest

TGF-β-related proteins are involved in a number of cellular processes.In embryogenesis, the 33 members of the TGF-β family of proteins areinvolved in regulating major developmental processes and the details ofthe formation of many organs. Much of this regulation occurs beforebirth; however, the family continues to regulate many processes afterbirth, including, but not limited to immune responses, wound healing,bone growth, endocrine functions and muscle mass. TGF-β-related proteinsare listed and described in U.S. Provisional Patent Applications61/722,919, filed Nov. 6, 2012; 61/722,969, filed Nov. 6, 2012 and61/823,552, filed May 15, 2013 the contents of each of which are hereinincorporated by reference in their entireties.

A list of exemplary TGF-β family pro-proteins, i.e. the protein afterremoval of the secretion signal sequence, is shown in Table 1. Thepro-protein contains, and is the precursor of, the prodomain and thegrowth factor. Shown in the Table are the names of the originating TGF-βfamily member and the pro-protein sequence. Also identified in “bold”and “underlined” are proprotein convertase cleavage sites. Uponcleavage, the resulting prodomain retains this site, whereas the maturegrowth factor begins following the cleavage site. It is noted thatLefty1 and Lefty2 are not cleaved by proprotein convertases just priorto the start of the mature growth factor.

TABLE 1 Pro-proteins of the TGF-beta family SEQ TGF MemberProdomain and growth factor Sequence ID NO TGF-β1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLA  1LYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSS RHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQL SNMIVRSCKCS TGF-β2SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVIS  2IYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLEIHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNR RKKR ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS TGF-β3SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVL  3ALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDEIHNPHLILMMIPPHRLDNPGQGGQ RKKR ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS GDF-11AEGPAAAAAAAAAAAAAGVGGE RSSR PAPSVAPEPDGCPVCVWRQHSREL  4RLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSL GPGAEGLHPFMELRVLENTKRSRR NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-8NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNIS  5 (myostatin)KDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPK RSRR DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS Inhibin-betaASPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLK  6KRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRR GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMI VEECGCS Inhibin-betaBSPTPPPTPAAPPPPPPPGSPGGSQDTCTSCGGFRRPEELGRVDGDFLEA  7VKRHILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGADGQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYVLEKGSRRKVRVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGERRLNLDVQCDSCQELAVVPVFVDPGEESHRPFVV VQARLGDSRHR IRKRGLECDGRTNLCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEGSCPAYLAGVPGSASSFHTAVVNQYRMRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA Inhibin-betaCTPRAGGQCPACGGPTLELESQRELLLDLAKRSILDKLHLTQRPTLNRPV  8SRAALRTALQHLHGVPQGALLEDNREQECEIISFAETGLSTINQTRLDFHFSSDRTAGDREVQQASLMFFVQLPSNTTWTLKVRVLVLGPHNTNLTLATQYLLEVDASGWHQLPLGPEAQAACSQGHLTLELVLEGQVAQSSVILGGAAH RPFVAARVRVGGKHQ IHRRGIDCQGGSRMCCRQEFFVDFREIGWHDWIIQPEGYAMNFCIGQCPLHIAGMPGIAASFHTAVLNLLKANTAAGTTGGGSCCVPTARRPLSLLYYDRDSNIVKTDIPDMVVEACGCS Inhibin-betaEQGTGSVCPSCGGSKLAPQAERALVLELAKQQILDGLHLTSRPRITHPPP  9QAALTRALRRLQPGSVAPGNGEEVISFATVTDSTSAYSSLLTFHLSTPRSHEILYHARLWLHVLPTLPGTLCLRIFRWGPRRRRQGSRTLLAEHHITNLGWHTLTLPSSGLRGEKSGVLKLQLDCRPLEGNSTVTGQPRRLLDTAGHQQP FLELKIRANEPGAG RARRRTPTCEPATPLCCRRDHYVDFQELGWRDWILQPEGYQLNYCSGQCPPHLAGSPGIAASFHSAVFSLLKANNPWPASTSCCVPTARRPLSLLYLDHNGNVVKTDVPDMVVEACGCS Lefty1LTGEQLLGSLLRQLQLKEVPTLDRADMEELVIPTHVRAQYVALLQRSHGD 10RSRGKRFSQSFREVAGRFLALEASTHLLVFGMEQRLPPNSELVQAVLRLFQEPVPKAALHRHGRLSPRSARARVTVEWLRVRDDGSNRTSLIDSRLVSVHESGWKAFDVTEAVNFWQQLSRPRQPLLLQVSVQREHLGPLASGAHKLVRFASQGAPAGLGEPQLELHTLDLGDYGAQGDCDPEAPMTEGTRCCRQEMYIDLQGMKWAENWVLEPPGFLAYECVGTCRQPPEALAFKWPFLGPRQCIASETDSLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP Lefty2LTEEQLLGSLLRQLQLSEVPVLDRADMEKLVIPAHVRAQYVVLLRRSHGD 11RSRGKRFSQSFREVAGRFLASEASTHLLVFGMEQRLPPNSELVQAVLRLFQEPVPKAALHRHGRLSPRSAQARVTVEWLRVRDDGSNRTSLIDSRLVSVHESGWKAFDVTEAVNFWQQLSRPRQPLLLQVSVQREHLGPLASGAHKLVRFASQGAPAGLGEPQLELHTLDLRDYGAQGDCDPEAPMTEGTRCCRQEMYIDLQGMKWAKNWVLEPPGFLAYECVGTCQQPPEALAFNWPFLGPRQCIASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP GDF-15LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTD 12LVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQ LELHLRPQAARGR RRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI Anti-MullerianLLGTEALRAEEPAVGTSGLIFREDLDWPPGIPQEPLCLVALGGDSNGSSS 13 hormonePLRVVGALSAYEQAFLGAVQRARWGPRDLATFGVCNTGDRQAALPSLRRLGAWLRDPGGQRLVVLHLEEVTWEPTPSLRFQEPPPGGAGPPELALLVLYPGPGPEVTVTRAGLPGAQSLCPSRDTRYLVLAVDRPAGAWRGSGLALTLQPRGEDSRLSTARLQALLFGDDHRCFTRMTPALLLLPRSEPAPLPAHGQLDTVPFPPPRPSAELEESPPSADPFLETLTRLVRALRVPPARASAPRLALDPDALAGFPQGLVNLSDPAALERLLDGEEPLLLLLRPTAATTGDPAPLHDPTSAPWATALARRVAAELQAAAAELRSLPGLPPATAPLLARLLALCPGGPGGLGDPLRALLLLKALQGLRVEWRGRDPRGPG RAQR SAGATAADGPCALRELSVDLRAERSVLIPETYQANNCQGVCGWPQSDRNPRYGNHVVLLLKMQVRGAALARPPCCVPTAYAGKLLISLSEERISAHHVPNMVATECGCR Inhibin-alphaCQGLELARELVLAKVRALFLDALGPPAVTREGGDPGVRRLPRRHALGGFT 14HRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFRPSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGHAPPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAH TRTRPPSGGE RARRSTPLMSWPWSPSALRLLQRPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPPTPAQPYSLLPGAQPCCAALPGTMRPLHVRTTSDGGYSFKYETVPNLLTQHCACI GDF-1PVPPGPAAALLQALGLRDEPQGAPRLRPVPPVMWRLFRRRDPQETRSGSR 15RTSPGVTLQPCHVEELGVAGNIVRHIPDRGAPTRASEPASAAGHCPEWTVVFDLSAVEPAERPSRARLELRFAAAAAAAPEGGWELSVAQAGQGAGADPGPVLLRQLVPALGPPVRAELLGAAWARNASWPRSLRLALALRPRAPAACAR LAEASLLLVTLDPRLCHPLARPRR DAEPVLGGGPGGAC RARR LYVSFREVGWHRWVIAPRGFLANYCQGQCALPVALSGSGGPPALNHAVLRALMHAAAPGAADLPCCVPARLSPISVLFFDNSDNVVLRQYEDMVVDECGCR GDF-3QEYVFLQFLGLDKAPSPQKFQPVPYILKKIFQDREAAATTGVSRDLCYVK 16ELGVRGNVLRFLPDQGFFLYPKKISQASSCLQKLLYFNLSAIKEREQLTLAQLGLDLGPNSYYNLGPELELALFLVQEPHVWGQTTPKPGKMFVLRSVPWPQGAVHFNLLDVAKDWNDNPRKNFGLFLEILVKEDRDSGVNFQPEDTCARLRCSLHASLLVVTLNPDQCHPS RKRR AAIPVPKLSCKNLCHRHQLFINFRDLGWHKWIIAPKGFMANYCHGECPFSLTISLNSSNYAFMQALMHAVDPEIPQAVCIPTKLSPISMLYQDNNDNVILRHYEDMVVDECGCG GDF-5APDLGQRPQGTRPGLAKAEAKERPPLARNVFRPGGHSYGGGATNANARAK 17GGTGQTGGLTQPKKDEPKKLPPRPGGPEPKPGHPPQTRQATARTVTPKGQLPGGKAPPKAGSVPSSFLLKKAREPGPPREPKEPFRPPPITPHEYMLSLYRTLSDADRKGGNSSVKLEAGLANTITSFIDKGQDDRGPVVRKQRYVFDISALEKDGLLGAELRILRKKPSDTAKPAAPGGGRAAQLKLSSCPSGRQPASLLDVRSVPGLDGSGWEVFDIWKLFRNFKNSAQLCLELEAWERGRAVDLRGLGFDRAARQVHEKALFLVFGRTKKRDLFFNEIKARSGQDDKTVYEYLFSQR RKRRAPLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR GDF-6FQQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREGQEPQPRPQ 18DEPRAQQPRAQEPPGRGPRVVPHEYMLSIYRTYSIAEKLGINASFFQSSKSANTITSFVDRGLDDLSHTPLRRQKYLFDVSMLSDKEELVGAELRLFRQAPSAPWGPPAGPLHVQLFPCLSPLLLDARTLDPQGAPPAGWEVFDVWQGLRHQPWKQLCLELRAAWGELDAGEAEARARGPQQPPPPDLRSLGFGRRVRPPQERALLVVFTRSQRKNLFAEMREQLGSAEAAGPGAGAEGSWPPPSGAPDA RPWLPSPGR RRRR TAFASRHGKR HG KKSR LRCSKKPLHVNFKELGWDDWIIAPLEYEAYHCEGVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCCVPTKLTPISILYIDAGNNVVYKQYEDMVVESCGCR GDF-7RDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAQAAGAAAVPAAAVPR 19ARAARRAAGSGFRNGSVVPHHFMMSLYRSLAGRAPAGAAAVSASGHGRADTITGFTDQATQDESAAETGQSFLFDVSSLNDADEVVGAELRVLRRGSPESGPGSWTSPPLLLLSTCPGAARAPRLLYSRAAEPLVGQRWEAFDVADAMRRHRREPRPPRAFCLLLRAVAGPVPSPLALRRLGFGWPGGGGSAAEERAVLVVSSRTQRKESLFREIRAQARALGAALASEPLPDPGTGTASPRAVIGGR RR RRTALAGTRTAQGSGGGAGRGHG RRGR SRCSRKPLHVDFKELGWDDWIIAPLDYEAYHCEGLCDFPLRSHLEPTNHAIIQTLLNSMAPDAAPASCCVPARLSPISILYIDAANNVVYKQYEDMVVEACGCR BMP-10SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKDEFLKTLNLSDI 20PTQDSAKVDPPEYMLELYNKFATDRTSMPSANIIRSFKNEDLFSQPVSFNGLRKYPLLFNVSIPHHEEVIMAELRLYTLVQRDRMIYDGVDRKITIFEVLESKGDNEGERNMLVLVSGEIYGTNSEWETFDVTDAIRRWQKSGSSTHQLEVHIESKHDEAEDASSGRLEIDTSAQNKHNPLLIVFSDDQSSDKERKEELNEMISHEQLPELDNLGLDSFSSGPGEEALLQMRSNIIYDSTA RIRR NAKGNYCKRTPLYIDFKEIGWDSWIIAPPGYEAYECRGVCNYPLAEHLTPTKHAIIQALVHLKNSQKASKACCVPTKLEPISILYLDKGVVTYKFKYEGMAVSEC GCR BMP-9 (GDF-2)KPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNL 21SGVPSQDKTRVEPPQYMIDLYNRYTSDKSTTPASNIVRSFSMEDAISITATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGSVVIYDVLDGTDAWDSATETKTFLVSQDIQDEGWETLEVSSAVKRWVRSDSTKSKNKLEVTVESHRKGCDTLDISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLA RRKR SAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMS VAECGCR Noda1TVATALLRTRGQPSSPSPLAYMLSLYRDPLPRADIIRSLQAEDVAVDGQN 22WTFAFDFSFLSQQEDLAWAELRLQLSSPVDLPTEGSLAIEIFHQPKPDTEQASDSCLERFQMDLFTVTLSQVTFSLGSMVLEVTRPLSKWLKRPGALEKQMSRVAGECWPRPPTPPATNVLLMLYSNLSQEQRQLGGSTLLWEAESSWRA QEGQLSWEWGK RHRRHHLPDRSQLCRKVKFQVDFNLIGWGSWITYPKQYNAYRCEGECPNPVGEEFHPTNHAYIQSLLKRYQPHRVPSTCCAPVKTKPLSMLYVDNGRVLLDHEIKDMIVEECGCL BMP-2LVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRPTPSRDA 23VVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEELPETSGKTTRRFFFNLSSIPTEEFITSAELQVFREQMQDALGNNSSFFIHRINIYEIIKPATANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVSKRHV RISR SLHQDEHSWSQIRPLLVTFGHDGK GHPLHK REKRQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR BMP-4GASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATLLQMFGLRR 24RPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSFHHEEHLENIPGTSENSAFRFLFNLSSIPENEVISSAELRLFREQVDQGPDWERGFHRINIYEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTHLHQTRTHQGQHV RISR SLPQGSGNWAQLR PLLVTFGHDGRGHALTRRRRAKR SPKHHSQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR BMP-5DNHVHSSFIYRRLRNHERREIQREILSILGLPHRPRPFSPGKQASSAPLF 25MLDLYNAMTNEENPEESEYSVRASLAEETRGARKGYPASPNGYPRRIQLSRTTPLTTQSPPLASLHDTNFLNDADMVMSFVNLVERDKDFSHQRRHYKEFRFDLTQIPHGEAVTAAEFRIYKDRSNNRFENETIKISIYQIIKEYTNRDADLFLLDTRKAQALDVGWLVFDITVTSNHWVINPQNNLGLQLCAETGDGRSINVKSAGLVGRQGPQSKQPFMVAFFKASEVLL RSVR AANKRKNQNRNKSSSHQDSSRMSSVGDYNTSEQKQACKKHELYVSFRDLGWQDWIIAPEGYAAFYCDGECSFPLNAHMNATNHAIVQTLVHLMFPDHVPKPCCAPTKLNAISVLYFDDSSNVILKKYRNMVVRSCGCH BMP-6CCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFLYRR 26LKTQEKREMQKEILSVLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLPRGEPPPGRLKSAPLFMLDLYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQDSAFLNDADMVMSFVNLVEYDKEFSPRQRHHKEFKFNLSQIPEGEVVTAAEFRIYKDCVMGSFKNQTFLISIYQVLQEHQHRDSDLFLLDTRVVWASEEGWLEFDITATSNLWVVTPQHNMGLQLSVVTRDGVHVHPRAAGLVGRDGPYDKQPFMVAFFKVSEVHV RTTR SAS SRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPKGYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH BMP-7DFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRPRPHLQGKHNSA 27PMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVA FFKATEVHF RSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACG CH BMP-8AGGGPGLRPPPGCPQRRLGA RERR DVQREILAVLGLPGRPRPRAPPAASRL 28PASAPLFMLDLYHAMAGDDDEDGAPAEQRLGRADLVMSFVNMVERDRALGHQEPHWKEFRFDLTQIPAGEAVTAAEFRIYKVPSIHLLNRTLHVSMFQVVQEQSNRESDLFFLDLQTLRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLYVETEDGHSVDPGLAGLLGQRAPRSQQPFVVTFFRASPSPI RTPR AVRPLRRRQPKKSNELPQANRLPGIFDDVRGSHGRQVCRRHELYVSFQDLGWLDWVIAPQGYSAYYCEGECSFPLDSCMNATNHAILQSLVHLMKPNAVPKACCAPTKLSATSVLYYDSSNNVILRKHRNMVVKACGCH BMP-8B GGGPGLRPPPGCPQRRLGA RERRDVQREILAVLGLPGRPRPRAPPAASRL 29PASAPLFMLDLYHAMAGDDDEDGAPAERRLGRADLVMSFVNMVERDRALGHQEPHWKEFRFDLTQIPAGEAVTAAEFRIYKVPSIHLLNRTLHVSMFQVVQEQSNRESDLFFLDLQTLRAGDEGWLVLDVTAASDCWLLKRHKDLGLRLYVETEDGHSVDPGLAGLLGQRAPRSQQPFVVTFFRASPSPI RTPR AVRPLRRRQPKKSNELPQANRLPGIFDDVHGSHGRQVCRRHELYVSFQDLGWLDWVIAPQGYSAYYCEGECSFPLDSCMNATNHAILQSLVHLMMPDAVPKACCAPTKLSATSVLYYDSSNNVILRKHRNMVVKACGCH BMP-15MEHRAQMAEGGQSSIALLAEAPTLPLIEELLEESPGEQPRKPRLLGHSLR 30YMLELYRRSADSHGHPRENRTIGATMVRLVKPLTSVARPHRGTWHIQILGFPLRPNRGLYQLVRATVVYRHHLQLTRFNLSCHVEPWVQKNPTNHFPSSEGDSSKPSLMSNAWKEMDITQLVQQRFWNNKGHRILRLRFMCQQQKDSGGLELWHGTSSLDIAFLLLYFNDTHKSIRKAKFLPRGMEEFMERESLL RRTR QADGISAEVTASSSKHSGPENNQCSLHPFQISFRQLGWDHWIIAPPFYTPNYCKGTCLRVLRDGLNSPNHAIIQNLINQLVDQSVPRPSCVPYKYVPISVLMIEANGSILYKEYEGMIAESCTCR GDF-9SQASGGEAQIAASAELESGAMPWSLLQHIDERDRAGLLPALFKVLSVGRG 31GSPRLQPDSRALHYMKKLYKTYATKEGIPKSNRSHLYNTVRLFTPCTRHKQAPGDQVTGILPSVELLFNLDRITTVEHLLKSVLLYNINNSVSFSSAVKCVCNLMIKEPKSSSRTLGRAPYSFTFNSQFEFGKKHKWIQIDVTSLLQPLVASNKRSIHMSINFTCMKDQLEHPSAQNGLFNMTLVSPSLILYLNDTSAQAYHSWYSLHYKRRPSQGPDQERSLSAYPVGEEAAEDGRSSHH RHRR GQETVSSELKKPLGPASFNLSEYFRQFLLPQNECELHDFRLSFSQLKWDNWIVAPHRYNPRYCKGDCPRAVGHRYGSPVHTMVQNIIYEKLDSSVPRPSCVPAKYSPLSVLTIEPDGSIAYKEYEDMIATKCTCR BMP-3ERPKPPFPELRKAVPGDRTAGGGPDSELQPQDKVSEHMLRLYDRYSTVQA 32ARTPGSLEGGSQPWRPRLLREGNTVRSFRAAAAETLERKGLYIFNLTSLTKSENILSATLYFCIGELGNISLSCPVSGGCSHHAQRKHIQIDLSAWTLKFSRNQSQLLGHLSVDMAKSHRDIMSWLSKDITQLLRKAKENEEFLIGFNITSKGRQLPKRRLPFPEPYILVYANDAAISEPESVVSSLQGHRNFPTGTVPKWDSHIRAALSIERRKKRSTGVLLPLQNNELPGAEYQYKKDEVWEERKPYK TLQAQAPEKSKNK KKQRKGPHRKSQTLQFDEQTLK KARR KQWIEPRNCARRYLKVDFADIGWSEWIISPKSFDAYYCSGACQFPMPKSLKPSNHATIQSIVRAVGVVPGIPEPCCVPEKMSSLSILFFDENKNVVLKVYPNMTVESCACR GDF-10SHRAPAWSALPAAADGLQGDRDLQRHPGDAAATLGPSAQDMVAVHMHRLY 33EKYSRQGARPGGGNTVRSFRARLEVVDQKAVYFFNLTSMQDSEMILTATFHFYSEPPRWPRALEVLCKPRAKNASGRPLPLGPPTRQHLLFRSLSQNTATQGLLRGAMALAPPPRGLWQAKDISPIVKAARRDGELLLSAQLDSEERDPGVPRPSPYAPYILVYANDLAISEPNSVAVTLQRYDPFPAGDPEPRAAPNNSADPRVRRAAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRALKPRPGRKDRRKKGQEVFMAASQVLDFDEKTMQ KARR KQWDEPRVCSRRYLKVDFADIGWNEWIISPKSFDAYYCAGACEFPMPKIVRPSNHATIQSIVRAVGIIPGIPEPCCVPDKMNSLGVLFLDENRNVVLKVYPNMSVDTCACR GDNFFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQ 34 ATIK RLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI NRTNIWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAQYRALLQGAPDAME 35LRELTPWAGRPPGPRRRAGP RRRR ARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEAAARVYDLGLRRLRQRRRLRRERVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV PSPNWGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPLARLRRALSGPCQLWSL 36TLSVAELGLGYASEEKVIFRYCAGSCPRGARTQHGLALARLQGQGRAHGGPCCRPTRYTDVAFLDDRHRWQRLPQLSAAACGCGG ARTNSLGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSG RARR PPPQPSRPA 37 PPPPAPPSALPRGGRAAR AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG

It is noted that some prodomains may be cleaved by proprotein convertaseenzymes. As used herein, the term “proprotein convertase” refers to anenzyme that cleaves a prodomain from a translated protein to facilitateprotein maturation. Some proprotein convertases of the present inventioninclude the subtilisin-like proprotein convertase (SPC) family memberenzymes. The SPC family comprises calcium-dependent serine endoproteasesthat include, but are not limited to furin/PACE, PC1/3, PC2, PC4, PC5/6,PACE4 and PC7 (Fuller et al., 2009. Invest Ophthalmol Vis Sci.50(12):5759-68, the contents of which are herein incorporated byreference in their entirety.) GDF-11 may in, in some cases, be cleavedby PC5/6. In some cases, proprotein convertases may cleave proproteinsat additional sites, other than those indicated in Table 1. In someembodiments, pro-proteins may be cleaved at a first cleavage site (thefirst site being the site closest to the N-terminus). In otherembodiments, pro-proteins may be cleaved at a cleavage site other than afirst cleavage site. In some cases, proprotein convertase cleavage mayoccur intracellularly. In some cases, proprotein convertase cleavage mayoccur extracellularly.

Many TGF-β family member proteins are synthesized in conjunction withprodomains. Some prodomains may remain associated with growth factorsafter cleavage. Such associations may form latent growthfactor-prodomain complexes (GPCs) that modulate the availability ofgrowth factors for cell signaling. Growth factors may be released fromlatency in GPCs through associations with one or more extracellularproteins. In some cases, growth factor release may rely on force appliedto GPCs through extracellular protein interactions. Such forces may pullfrom C-terminal and/or N-terminal regions of GPCs resulting in therelease of associated growth factors.

In some TGF-β family members, the prodomain portion of the GPC isresponsible for growth factor retention and blocking the interaction ofretained growth factors with their receptors. Prodomain portions of GPCsthat function in this regard are referred to as latency associatedpeptides (LAPs). TGF-β1, 2 and 3 are know to comprise LAPs. Someprodomains may comprise LAP-like domains. As used herein, the term“LAP-like domain” refers to prodomain portions of GPCs and/or freeprodomains that may be structurally similar or synthesized in a similarmanner to LAPs, but that may not function to prevent growthfactor/receptor interactions. GDF-8 and GDF-11 prodomains compriseLAP-like domains.

Depending on a variety of factors, growth factors may be free orassociated with one or more LAP or LAP-like domains. FIG. 3 is aschematic depicting an embodiment wherein a growth factor dimer mayassociate with a LAP dimer. In some embodiments, GPCs comprise proteinmodules necessary for different aspects of growth factor signaling,secretion, latency and/or release from latent GPCs. As used herein, theterm “protein module” refers to any component, region and/or feature ofa protein. Protein modules may vary in length, comprising one or moreamino acids. Protein modules may be from about 2 amino acid residues inlength to about 50 amino acid residues in length, from about 5 aminoacid residues in length to about 75 amino acid residues in length, fromabout 10 amino acid residues in length to about 100 amino acid residuesin length, from about 25 amino acid residues in length to about 150amino acid residues in length, from about 125 amino acid residues inlength to about 250 amino acid residues in length, from about 175 aminoacid residues in length to about 400 amino acid residues in length, fromabout 200 amino acid residues in length to about 500 amino acid residuesin length and/or at least 500 amino acid residues in length.

In some embodiments, protein modules comprise one or more regions withknown functional features (e.g. protein binding domain, nucleic acidbinding domain, hydrophobic pocket, etc.) Protein modules may comprisefunctional protein domains necessary for different aspects of growthfactor signaling, secretion, latency and/or release from latentconformations.

In some embodiments, protein modules may be derived from TGF-β-relatedproteins. Such protein modules may include, but are not limited tolatency-associated peptides (LAPs), LAP-like domains, growth factordomains, fastener regions, proprotein convertase cleavage sites (e.g.furin cleavage sites), B/TP cleavage sites, arm regions, finger regions,residues (such as cysteine residues for example) for extracellularprotein [e.g. latent TGF-β binding protein (LTBP), fibrillin and/orglycoprotein A repetitions predominant (GARP) protein] associations,latency loops (also referred to herein as latency lassos) alpha 1helical regions, alpha 2 helical regions, RGD sequences and bowtieregions. FIG. 4 is a schematic diagram of an embodiment depicting LAPand growth factor dimers comprising protein modules.

In some embodiments, protein modules may be derived from one or moreTGF-β isoform (e.g. TGF-β1, TGF-β2 and/or TGF-β3). Such protein modulesmay comprise the protein modules and/or amino acid sequences listed inTable 2. Some protein modules of the present invention may compriseamino acid sequences similar to those in Table 2, but compriseadditional or fewer amino acids than those listed. Such amino acidsequences may comprise about 1 more or fewer amino acids, about 2 moreor fewer amino acids, about 3 more or fewer amino acids, about 4 more orfewer amino acids, about 5 more or fewer amino acids, about 6 more orfewer amino acids, about 7 more or fewer amino acids, about 8 more orfewer amino acids, about 9 more or fewer amino acids, about 10 more orfewer amino acids or greater than 10 more or fewer amino acids onN-terminal and/or C-terminal ends.

TABLE 2 TGF-β protein modules TGF-β Family SEQ Member Protein ModuleProdomain and growth factor Sequence ID NO TGF-β1 latency associatedLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 38 peptideGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRG DLATIHGMNRPFLLLMATPLERAQHLQSSRHRRTGF-β2 latency associated SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 39peptide DYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTR KKNSGKTPHLLLMLLPSYRLESQQTNRRKKRTGF-β3 latency associated SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 40peptide PEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKD HHNPHLILMMIPPHRLDNPGQGGQRKKR TGF-β1straight jacket LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 41 region GEVPPGPLPTGF-β2 straight jacket SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 42 regionDYPEPEEVP TGF-β3 straight jacket SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 43region PEPTVMTHVP TGF-β1 growth factorALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP 44 domainKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRS CKCS TGF-β2 growth factorALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEP 45 domainKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKS CKCS TGF-β3 growth factorALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEP 46 domainKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKS CKCS TGF-β1 fastener regionresidues 74-76, YYA — TGF-β2 fastener region residues 79-81, YYA —TGF-β3 fastener region residues 80-82, YYA — TGF-β1 furin cleavage siteRHRR 47 region TGF-β2 furin cleavage site RKKR 48 region TGF-β3furin cleavage site RKKR 48 region TGF-β1 arm regionEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTR 49VLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMN RPFLLLMATPLERAQHLQSSRHRR TGF-β2arm region PEVISIYNSTRDLLQEKASRRAAACERERSDEEYYA 50KEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPH LLLMLLPSYRLESQQTNRRKKR TGF-β3arm region YQVLALYNSTRELLEEMHGEREEGCTQENTESEYYA 51KEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMM IPPHRLDNPGQGGQRKKR TGF-β1fingers region 1 CVRQLYIDFRKDLGWKWIHEPKGYHANFC 52 TGF-β2fingers region 1 CLRPLYIDFKRDLGWKWIHEPKGYNANFCA 53 TGF-β3fingers region 1 CVRPLYIDFRQDLGWKWVHEPKGYYANFCS 54 TGF-β1fingers region 2 CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 55 TGF-β2fingers region 2 CVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS 56 TGF-β3fingers region 2 CVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS 57 TGF-β1residue for LTBP Cys 4 — association TGF-β2 residue for LTBP Cys 5 —association TGF-β3 residue for LTBP Cys 7 — association TGF-β1residue for GARP Cys 4 — association TGF-β2 residue for GARP Cys 5 —association TGF-β3 residue for GARP Cys 7 — association TGF-β1latency loop LASPPSQGEVPPGPL 58 TGF-β2 latency loop LTSPPEDYPEPEE 59TGF-β3 latency loop LTSPPEPTVMTHV 60 TGF-β1 alpha 1 helicalLSTCKTIDMELVKRKRIEAIRGQILSKLR 61 region TGF-β2 alpha 1 helicalLSTCSTLDMDQFMRKRIEAIRGQILSKLK 62 region TGF-β3 alpha 1 helicalLSLSTCTTLDFGHIKKKRVEAIRGQILSKLR 63 region TGF-β1 trigger loop regionNGFTTGRRGDLATIHGMNRP 64 TGF-β2 trigger loop regionFAGIDGTSTYTSGDQKTIKSTRKKNSGKTP 65 (long) TGF-β3 trigger loop regionGVDNEDDHGRGDLGRLKKQKDHHNP 66 TGF-β1 RGD sequence residue 215-217, RGD —region TGF-β3 RGD sequence residue 241-243, RGD — region TGF-β1bowtie region CSCDSRDNTLQVD 67 TGF-β2 bowtie regionCPCCTFVPSNNYIIPNKSEELEAR 68 TGF-β3 bowtie region CPCHTFQPNGDILENIHEVMEIK69

In some embodiments, LAPs or LAP-like domains comprise the prodomainportion of a TGF-β-related protein and/or GPC. Some LAPs or LAP-likedomains may associate with growth factors in GPCs. Some LAPs maysterically prevent growth factor association with one or more cellularreceptors. LAPs or LAP-like domains may comprise arm regions and/orstraight jacket regions. Some LAP or LAP-like domains may compriseC-terminal regions referred to herein as “bowtie regions.” In some LAPor LAP-like domain dimers, bowtie regions of each monomer may associateand/or interact. Such associations may comprise disulfide bondformation, as is found between monomers of TGF-β isoform LAPs.

In some embodiments, arm regions may comprise trigger loop regions.Trigger loops may comprise regions that associate with integrins. Suchregions may comprise amino acid sequences comprising RGD (Arg-Gly-Asp).Regions comprising RGD sequences are referred to herein as RGD sequenceregions. In some embodiments, LAPs or LAP-like domains comprise latencyloops (also referred to herein as latency lassos). Some latency loopsmay maintain associations between LAPs or LAP-like domains and growthfactors present within GPCs. LAPs or LAP-like domains may also comprisefastener regions. Such fastener regions may maintain associationsbetween LAPs or LAP-like domains and growth factors present within GPCs.Some fastener regions may maintain LAP or LAP-like domain conformationsthat promote growth factor retention.

In some cases, GPCs may require enzymatic cleavage for dissociation ofbound growth factors. Such cleavage may be carried out in some instancesby members of the BMP-1/Tolloid-like proteinase (B/TP) family (Muir etal., 2011. J Biol Chem. 286(49):41905-11, the contents of which areherein incorporated by reference in their entirety.) Thesemetaloproteinases may include, but are not limited to BMP-1, mammaliantolloid protein (mTLD) mammalian tolloid-like 1 (mTLL1) and mammaliantolloid-like 2 (mTLL2.) Exemplary GPCs that may be cleaved by suchmetalloproteinases may include, but are not limited to GDF-8 and GDF-11.In some cases, GDF-8 may be cleaved by mTLL2. In some cases, tolloidcleavage may occur intracellularly. In some cases, tolloid cleavage mayoccur extracellularly.

Straight jacket regions may comprise alpha 1 helical regions. In someembodiments, alpha 1 helical regions may be positioned between growthfactor monomers. Some alpha 1 helical regions comprise N-terminalregions of LAPs or LAP-like domains. Alpha 1 helical regions may alsocomprise N-terminal regions for extracellular associations. Suchextracellular associations may comprise extracellular matrix proteinsand/or proteins associated with the extracellular matrix. Someextracellular associations may comprise associations with proteins thatmay include, but are not limited to LTBPs (e.g. LTBP1, LTBP2, LTBP3and/or LTBP4), fibrillins (e.g. fibrillin-1, fibrillin-2, fibrillin-3and/or fibrillin-4) perlecan, decorin and/or GARPs (e.g. GARP and/orLRRC33). N-terminal extracellular associations may comprise disulfidebonds between cysteine residues. In some cases, extracellular matrixproteins and/or proteins associated with the extraceullar matrix maycomprise bonds with one or more regions of LAPs/LAP-like domains otherthan N-terminal regions.

In some embodiments, growth factor domains comprise one or more growthfactor monomers. Some growth factor domains comprise growth factordimers. Such growth factor domains may comprise growth factor homodimersor heterodimers (comprising growth factor monomers from differentTGF-β-related proteins.) Some growth factor domains may comprise fingersregions. Such fingers regions may comprise β-pleated sheets. Fingersregions may associate with LAPs or LAP-like domains. Some fingersregions may maintain association between growth factor domains and LAPsor LAP-like domains.

In some embodiments, recombinant proteins of the present invention maycomprise protein modules from growth differentiation factor (GDF)proteins. Such GDF protein modules may comprise the protein modulesand/or amino acid sequences listed in Table 3. In some embodiments,protein modules of the present invention may comprise amino acidsequences similar to those in Table 3, but comprise additional or feweramino acids than those listed. Some such amino acid sequences maycomprise about 1 more or fewer amino acids, about 2 more or fewer aminoacids, about 3 more or fewer amino acids, about 4 more or fewer aminoacids, about 5 more or fewer amino acids, about 6 more or fewer aminoacids, about 7 more or fewer amino acids, about 8 more or fewer aminoacids, about 9 more or fewer amino acids, about 10 more or fewer aminoacids or greater than 10 more or fewer amino acids on N-terminal and/orC-terminal ends.

TABLE 3 GDF protein modules TGF-β Family SEQ  Member Protein ModuleProdomain and growth factor Sequence ID NO GDF-8 prodomainNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQ 70ILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAV TFPGPGEDGLNPFLEVKVTDTPKRSRR GDF-11prodomain AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 71GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGP GAEGLHPFMELRVLENTKRSRR GDF-8straight jacket NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQ 72 regionILSKLRLETAPNISKDVIRQLLPKAPPL GDF-11 straight jacketAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 73 regionGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNIS REVVKQLLPKAPPL GDF-8 growth factorDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPK 74 domainRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGC S GDF-11 growth factorNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPK 75 domainRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGC S GDF-8 fastener regionresidues 87-89, DYH — GDF-11 fastener region residues 110-112, EYH —GDF-8 furin cleavage site RSRR 76 region GDF-11 furin cleavage site RSRR76 region GDF-8 BMP/Tolloid between residues R75 and D76 — cleavage siteGDF-11 BMP/Tolloid between residues G97 and D98 — cleavage site GDF-8arm region RELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPT 77ESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALD ENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRGDF-11 arm region QQILDLHDFQGDALQPEDFLEEDEYHATTETVISMA 78QETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVL ENTKRSRR GDF-8 fingers region 1CRYPLTVDFEAFGWDWIIAPKRYKANYCS 79 GDF-11 fingers region 1CRYPLTVDFEAFGWDWIIAPKRYKANYCS 79 GDF-8 fingers region 2CTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS 80 GDF-11 fingers region 2CTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS 81 GDF-8 latency loopRLETAPNISKDVIRQLLPKAPPL 82 GDF-11 latency loop RLKEAPNISREVVKQLLPKAPP 83GDF-8 alpha 1 helical GLCNACTWRQNTKSSRIEAIKIQIL SK 84 region GDF-11alpha 1 helical DGCPVCVWRQHSRELRLESIKSQILSKL 85 region GDF-8bowtie region DENGHDLAVTFPGP 86 GDF-11 bowtie region DPSGTDLAVTSLG 87

Some recombinant proteins of the present invention may comprise GDF-15,GDF-15 signaling pathway-related proteins and/or modules and/or portionsthereof. GDF-15 is a TGF-β family protein that is highly expressed inliver. Expression of GDF-15 is dramatically upregulated following liverinjury (Hsiao et al. 2000. Mol Cell Biol. 20(10):3742-51.) Additionally,its expression in macrophages may serve a protective function in thecontext of atherosclerosis, possibly through regulation of adhesionmolecule expression (Preusch et al., 2013. Eur J Med Res. 18:19.) Whilea member of the TGF-β family, GDF-15 comprises less than 30% homologywith other members, making it the most divergent member of the family(Tanno et al., 2010. Curr Opin Hematol. 17(3):184-90, the contents ofwhich are incorporated herein by reference in their entirety.) Themature form is soluble and can be found in the blood stream.Interestingly, GDF-15 levels in circulation have been found tonegatively correlate with hepcidin levels, suggesting a role for GDF-15in iron load and/or metabolism (Finkenstedt et al., 2008. BritishJournal of Haematology. 144:789-93.) Elevated GDF-15 in the blood isalso associated with ineffective and/or apoptotic erythropoiesis, suchas in subjects suffering from beta-thalassemia or dyserythropoieticanemias.

In some embodiments, recombinant proteins of the present invention maycomprise protein modules from activin subunits. Such protein modules maycomprise the protein modules and/or amino acid sequences of the activinsubunit inhibin beta A, listed in Table 4. In some embodiments, proteinmodules of the present invention may comprise amino acid sequencessimilar to those in Table 4, but comprise additional or fewer aminoacids than those listed. Some such amino acid sequences may compriseabout 1 more or fewer amino acids, about 2 more or fewer amino acids,about 3 more or fewer amino acids, about 4 more or fewer amino acids,about 5 more or fewer amino acids, about 6 more or fewer amino acids,about 7 more or fewer amino acids, about 8 more or fewer amino acids,about 9 more or fewer amino acids, about 10 more or fewer amino acids orgreater than 10 more or fewer amino acids on N-terminal and/orC-terminal ends.

TABLE 4 Inhibin beta Aprotein modules SEQ Protein ModuleProdomain and growth factor Sequence ID NO latency associatedSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVK 88 peptide (LAP)KHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFL MLQARQSEDHPHRRRRRstraight jacket SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVK 89 regionKHILNMLHLKKRPDVTQPVPKAALLN growth factorRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHAN 90 domainYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS fastener regionresidues 89-91, RRA -- furin cleavage site RRRR 91 region arm regionLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSE 92IITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGG AGADEEKEQSHRPFLMLQARQSEDHPHRRRRRfingers region 1 KKQFFVSFKDIGWNDWIIAPSGYHANYC 93 fingers region 2CVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS 94 latency loop LKKRPDVTQPVPKAALL95 alpha 1 helical ALAALPKDVPNSQPEMVEAVKKHILNML 96 region bowtie regionQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAG 97

Growth factor domains among TGF-β family members are more highlyconserved while prodomains comprise a much lower percent identity amongfamily members (FIG. 9.) Table 5 demonstrates this trend among TGF-βisoforms.

TABLE 5 Percent identity among TGF-β isoforms: LAP vs growth factorTGF-β1 TGF-β2 TGF-β3 TGF-β1 — 31.2% vs 71.2% 31.9% vs 76.7% TGF-β2 31.2%vs 71.2% — 44.4% vs 79.4% TGF-β3 31.9% vs 76.7% 44.4% vs 79.4% —

Prodomains may vary in length from about 50 to about 200, from about 100to about 400 or from about 300 to about 500 amino acids residues. Insome embodiments, prodomains range from about 169 to about 433 residues.Prodomains may be unrelated in sequence and/or low in homology. Someprodomains may have similar folds and/or three dimensional structures.Prodomains of TGF-β family members may comprise latency loops. Suchloops may be proline-rich. Latency loop length may determine the abilityof such loops to encircle growth factor finger regions.

In some embodiments, protein modules from some TGF-β family memberscomprise low sequence identity with protein modules from other TGF-βfamily members. Such low sequence identity may indicate specializedroles for such family members with distinct protein modules.

Association of GPCs with extracellular proteins may strengthenprodomain-growth factor interactions. In some embodiments, suchextracellular proteins may include, but are not limited to LTBPs,fibrillins and/or GARP. In some cases, extracellular proteinassociations are required to keep growth factors latent in GPCs.

GARP expression has been shown to be required for surface expression ofGPCs on the surface of cells of hematopoietic origin (Tran, D. Q. etal., GARP (LRRC32) is essential for the surface expression of latentTGF-β on platelets and activated FOXP3+ regulatory T cells. PNAS. 2009,Jun. 2. 106(32):13445-50.) GARP may act as a tether to hold GPCs inplace on the surface of these cells, including, but not limited toregulatory T-cells and/or platelets.

In some embodiments, recombinant proteins of the present invention maycomprise bone morphogenetic proteins (BMPs), a family of TGF-β-relatedproteins. Protein modules comprising sequences from BMPs may comprisesequences from any of those BMP modules disclosed in FIGS. 8A-8G. Whilerelated to other TGF-β family member proteins, BMPs generally signalthrough SMAD1, 5 and 8 proteins while TGF-β isoforms (e.g. TGF-β1,TGF-β2 and TGF-β3) signal through SMAD2 and SMAD3.

Some BMP receptors and/or co-receptors are also distinct from otherTGF-β family member proteins. Among these is the repulsive guidancemolecule (RGM) family of proteins. RGM proteins act as co-receptors forBMP signaling. There are three RGM family members, RGMA, RGMB and RGMC[also known as hemojuvelin (Hjv.)] Recombinant proteins of the presentinvention comprising one or more BMP protein module may be useful forthe development of antibodies and/or assays to study, enhance and/orperturb BMP interactions with RGM proteins.

Another family of GDF/BMP interacting proteins is C-terminal cysteineknot-like (CTCK) domain-containing proteins. In some cases, CTCKdomain-containing proteins may act antagonistically with regard toGDF/BMP signal transduction. CTCK domain-containing proteins include,but are not limited to Cerberus, Connective tissue growth factor (CTGF),DAN domain family member 5 (DAND5), Gremlin-1 (GREM1), Gremlin-2(GREM2), Mucin-19 (MUC19), Mucin-2 (MUC2), Mucin-5AC (MUC5AC), Mucin-5B(MUC5B), Mucin-6 (MUC6), Neuroblastoma suppressor of tumorigenicity 1(NBL1), Norrin (NDP), Otogelin (OTOG), Otogelin-like protein (OTOGL),Protein CYR61 (CYR61), Protein NOV homolog (NOV), Sclerostin (SOST),Sclerostin domain-containing protein 1 (SOSTDC1), SCO-spondin (SSPO),Slit homolog 1 protein (SLIT1), Slit homolog 2 protein (SLIT2), Slithomolog 3 protein (SLIT3), von Willebrand factor (VWF),WNT1-inducible-signaling pathway protein 1 (WISP1) andWNT1-inducible-signaling pathway protein 3 (WISP3).

Recombinant Proteins

In some embodiments, the present invention provides recombinantproteins. As used herein, the term “recombinant protein” refers to aprotein produced by an artificial gene and/or process (e.g. geneticengineering). Such recombinant proteins may comprise one or more proteinmodules from one or more TGF-β-related proteins. Some recombinantproteins disclosed herein may be useful as recombinant antigens. As usedherein, the term “recombinant antigen” refers to a recombinant proteinthat may be used to immunize one or more hosts for the production ofantibodies directed toward one or more epitopes present on suchrecombinant antigens. Some recombinant antigens may be cell-basedantigens. As used herein, the term “cell-based antigen” refers torecombinant antigens that are expressed in cells for presentation ofsuch antigens on the cell surface. Such cells may be used to immunizehosts for the production of antibodies directed to such cell-basedantigens.

In some embodiments, recombinant proteins disclosed herein may be usedas therapeutics. Recombinant proteins disclosed herein may modulategrowth factor (e.g. growth factors comprising TGF-β-related proteins)levels and/or activity (e.g. signaling) upon administration and/orintroduction to one or more subjects and/or niches.

In some embodiments, recombinant proteins disclosed herein may be usedto assay growth factor (e.g. growth factors comprising TGF-β-relatedproteins) levels and/or activity (e.g. signaling). Some recombinantproteins disclosed herein may be used in the isolation of antibodiesdirected to TGF-β-related proteins. Recombinant proteins of the presentinvention may also be used as recombinant antigens in the development ofstabilizing [reducing or preventing dissociation between two agents,(e.g. growth-factor release from GPCs, GPC release from one or moreprotein interactions)] and/or releasing [enhancing the dissociationbetween two agents (e.g. growth-factor release from GPCs, GPC releasefrom one or more protein interactions)] antibodies. Recombinant proteinsof the present invention may include TGF-β family member proteins aswell as components and/or protein modules thereof. Some recombinantproteins of the present invention may comprise prodomains withoutassociated growth factors, furin cleavage-deficient mutants, mutantsdeficient in extracellular protein associations and/or combinationsthereof.

In some embodiments, recombinant proteins may comprise detectablelabels. Detectable labels may be used to allow for detection and/orisolation of recombinant proteins. Some detectable labels may comprisebiotin labels, polyhistidine tags and/or flag tags. Such tags may beused to isolate tagged proteins. Proteins produced may compriseadditional amino acids encoding one or more 3C protease cleavage site.Such sites allow for cleavage at the 3C protease cleavage site upontreatment with 3C protease, including, but not limited to rhinovirus 3Cprotease. Such cleavage sites are introduced to allow for removal ofdetectable labels from recombinant proteins.

Recombinant GPCs

FIG. 5 is a schematic depicting an embodiment of a recombinant GPC.Recombinant proteins according to FIG. 5 comprising TGF-β-family memberproteins may comprise features including, but not limited to C-terminalregions of the mature growth factor, N-terminal regions of the prodomainand/or proprotein cleavage sites. The proprotein cleavage site ofrecombinant TGF-β GPCs may, for example, comprise the furin consensussequence RXXR wherein R is arginine and X indicates amino acid residuesthat may vary among TGF-β family members. Furin cleavage site sequences(although not limited to cleavage by furin alone and may includecleavage by other proprotein convertase enzymes) for each TGF-β familymember are indicated in Table 1. Recombinant GPCs according to theembodiment depicted in FIG. 5 may also comprise one or more cysteineresidues within and/or near the N-terminal region of the prodomain. Suchcysteine residues may be from about 1 to about 10 amino acids, fromabout 4 to about 15 amino acids, from about 5 to about 20 amino acidsand/or from about 7 to about 50 amino acids from the N-terminus of theprodomain. Recombinant GPCs may also comprise detectable labels. Suchdetectable labels may be useful for detection and/or isolation ofrecombinant GPCs. Detectable labels may comprise 2 or more histidine(His) residues. Such detectable labels may also be referred to herein aspolyhistidine tags. Polyhistidine tags may include hexa histidine tags(SEQ ID NO: 295) or HIS-TAG™ (EMD Biosciences, Darmstadt, Germany)comprising a chain of six histidine residues (SEQ ID NO: 295). Somepolyhistidine tags may be present at the N-terminus of recombinantproteins disclosed herein. Some polyhistidine tags may be present at theC-terminus of recombinant proteins disclosed herein. Proteins producedmay comprise additional amino acids encoding one or more 3C proteasecleavage site. Such sites allow for cleavage at the 3C protease cleavagesite upon treatment with 3C protease, including, but not limited torhinovirus 3C protease. Some cleavage sites may be introduced to allowfor removal of detectable labels from recombinant proteins.

In some embodiments of the present invention, recombinant GPCs maycomprise mutations in one or more amino acids as compared to wild typesequences. In some cases, one or more regions of proteolytic processingmay be mutated. Such regions may comprise proprotein convertase cleavagesites. Proprotein convertase (e.g. furin) cleavage site mutationsprevent enzymatic cleavage at that site and/or prevent enzymaticcleavage of growth factors from their prodomains (see FIG. 6.) Someproprotein convertase cleavage sites comprising RXXR sequences may bemutated to RXG (wherein X indicates a site where amino acid residues maybe variable). Such mutations are herein abbreviated as “D2G” mutationsand may be resistant to enzymatic cleavage. In some embodiments, furincleavage sites comprising RXXR sequences are mutated to AXXA. Such AXXAsequences may also be resistant to enzymatic cleavage.

In some embodiments, regions of proteolytic processing by tolloid and/ortolloid-like proteins may be mutated to prevent such proteolyticprocessing. In some embodiments, tolloid processing regions on GDF-8and/or GDF-11 may be mutated. In some embodiments, mutation of asparticacid residues to alanine residues within tolloid processing regionsprevents tolloid processing. Mutation of aspartic acid residue 76 (D76)of the GDF-8 (myostatin) proprotein has been shown to preventproteolytic activation of latent GDF-8 (Wolfman, N. M. et al., PNAS.2003, Oct. 6. 100(26):15842-6.) In some embodiments, Asp 120 (D120,residue number counted from the translated protein, D98 from theproprotein of SEQ ID NO: 4) in GDF-11 may be mutated to prevent tolloidprocessing (Ge et al., 2005. Mol Cell Biol. 25(14):5846-58, the contentsof which are herein incorporated by reference in their entirety.)

In some embodiments, one or more amino acids may be mutated in order toform recombinant GPCs with reduced latency. Such mutations are referredto herein as “activating mutations.” These mutations may introduce oneor more regions of steric clash between complex prodomains and growthfactor domains. As used herein, the term “steric clash,” when referringto the interaction between two proteins or between two domains and/orepitopes within the same protein, refers to a repulsive interactionbetween such proteins, domains and/or epitopes due to overlappingposition in three-dimensional space. Steric clash within GPCs may reducethe affinity between prodomains and growth factor domains, resulting inelevated ratios of free growth factor to latent growth factor. In someembodiments, one or more amino acids may be mutated in order to formrecombinant GPCs with increased latency. Such mutations are referred toherein as “stabilizing mutations.” These mutations may increase theaffinity between prodomains and growth factor domains, resulting indecreased ratios of free growth factor to latent growth factor.

In some embodiments, recombinant proteins of the present invention maycomprise any of the sequences listed in Table 6 or fragments thereof.

TABLE 6 Recombinant proteins SEQ Protein Sequence ID NO proTGF-β1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP   1LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCSproTGF-β1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP  98LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCSproTGF-β1 C4S LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP  99 (LAP)LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR proTGF-β1 D2GLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 100LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCSproTGF-β1 C4S D2G LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 101LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCSproTGF-β1 LAP LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP  38LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR proTGF-β2SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE   2VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNM IVKSCKCS proTGF-β2 C5SSLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 102VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNM IVKSCKCS pro TGF-β2 LAP C5SSLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 103VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKKRpro TGF-β2 C5S D2G SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 104VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS proTGF-β2 D2GSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 105VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKGALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS proTGF-β2 LAPSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE  39VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGK TPHLLLMLLPSYRLESQQTNRRKKRproTGF-β3 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT   3HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVV KSCKCS proTGF-β3 C7SSLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 106HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVV KSCKCS proTGF-β3 LAP C7SSLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 107HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPH LILMMIPPHRLDNPGQGGQRKKRproTGF-β3 C7S D2G SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 108HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPHLILMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVK SCKCS proTGF-β3 D2GSLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT 109HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPHLILMMIPPHRLDNPGQGGQRKGALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVK SCKCS proTGF-β3 LAPSLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMT  40HVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHEINPH LILMMIPPHRLDNPGQGGQRKKR

In some embodiments, activating mutations may comprise residues criticalfor LAP or LAP-like protein dimerization. Some activating mutations maycomprise TGF-β isoforms (TGF-β1, TGF-β2 and/or TGF-β3). Mutant GPCs withactivating mutations may comprise mutations that correspond to mutationsidentified in Camurati-Engelmann disease (CED). Subjects suffering fromCED typically have genetic defects in TGF-β1. Mutations identified insuch subjects include, but are not limited to mutations in residues Y81,R218, H222, C223 and C225. Residues C223 and C225 are necessary fordisulfide bond formation in LAP dimerization. Mutations to R218, H222,C223 and/or C225 may lead to weakened or disrupted disulfide bondformation and LAP dimerization. In some embodiments, CED mutations leadto elevated release of TGF-β and/or increased TGF-β activity. In someembodiments, recombinant GPCs comprising TGF-β1 with CED mutationscomprise sequences listed in Table 7. The amino acid substitutionsindicated in these proteins reflect the residue number as counted fromthe start of the translated protein (before removal of the secretionsignal sequence).

TABLE 7 Recombinant GPCs with Camurati-Engelmann mutations SEQ IDProtein Sequence NO proTGF-β1 Y81HLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 110LPEAVLALHNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF-β1 R218CLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 111LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFCLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF-β1 H222DLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 112LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSADCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF-β1 C223RLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 113LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHRSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF-β1 C225RLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 114LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS proTGF-β1 C223RLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP 115 C225RLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHRSRDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

GPCs comprising CED mutations may find several uses in the context ofthe present invention. In some embodiments, such GPCs may be used toproduce recombinant proteins comprising LAPs or LAP-like domainscomplexed with GARP. Coexpression of the entire GPC with GARP may benecessary in some embodiments, for proper association and folding.Through expression of GPCs comprising CED mutations, growth factors maybe able to dissociate leaving the desired GARP-LAP complex. Y81Hmutations may be useful in this regard. Y81H mutations lead to growthfactor release, but do not disrupt disulfide bonding between LAPmonomers at residues C223 and C225. Therefore, GARP-LAP complexes formedthrough expression of Y81H GPC mutants may comprise intact LAP dimerswherein growth factors have become dissociated. In some embodiments,additional co-expression or addition of excess furin during theproduction process may enhance growth factor dissociation as well.

GPCs comprising CED mutations may be expressed to allow for theproduction and release of mature growth factor. Some GPC-free growthfactors expressed according to this method may be used to assessantibody reactivity, for example in enzyme-linked immunosorbent assays(ELISAs.) Some GPCs comprising CED mutations may be expressed to allowfor the production and release of GPC-bound growth factors. GPCscomprising CED mutations may be expressed to allow for the productionand release of chimeric proteins comprising the TGF-β1 LAP (or proteinmodules or fragments thereof) expressed with one or more protein modulesfrom other TGF-β family members. Such chimeric proteins may compriseTGF-β1 LAP and TGF-β2 or TGF-β3 growth factor domains.

Furin cleavage of recombinant proteins of the invention may in somecases occur intracellularly. In some cases furin cleavage of recombinantproteins of the invention may occur extracellularly.

In some embodiments, recombinant GPCs of the present invention maycomprise mutations in one or more N-terminal regions for extracellularassociations. As used herein, the term “N-terminal region forextracellular association” refers to regions at or near proteinN-termini that may be necessary for extracellular associations with oneor more N-terminal regions. Such regions may comprise at least the firstN-terminal residue, at least the first 5 N-terminal residues, at leastthe first 10 N-terminal residues, at least the first 20 amino acidresidues and/or at least the first 50 amino acid residues. Somemutations may comprise from about 1 amino acid residue to about 30 aminoacid residues, from about 5 amino acid residues to about 40 amino acidresidues and/or from about 10 amino acid residues to about 50 amino acidresidues at or near protein N-termini. Such regions may compriseresidues for LTBP, fibrillin and/or GARP association. In some cases, oneor more cysteine residues present within and/or near N-terminal regionsfor extracellular associations may be necessary for such associations.In some embodiments, cysteine residues present within and/or nearN-terminal regions for extracellular associations are present withinabout the first 2 N-terminal residues, about the first 3 N-terminalresidues, about the first 4 N-terminal residues, about the first 5N-terminal residues, about the first 6 N-terminal residues, about thefirst 7 N-terminal residues and/or at least the first 30 N-terminalresidues. Some mutations in one or more N-terminal regions forextracellular associations comprise substitution and/or deletion of suchcysteine residues. Such mutations may modulate the association of GPCsand/or prodomains with one or more extracellular proteins, including,but not limited to LTBPs, fibrillins and/or GARP. These mutations mayalso comprise substitution of one or more cysteine with another aminoacid. Cysteine residue substitutions are abbreviated herein as “C#X”wherein # represents the residue number [counting from the N-terminus ofthe pro-protein (without the signal peptide)] of the original cysteineresidue and X represents the one letter amino acid code for the aminoacid that is used for substitution. Any amino acid may be used for suchsubstitutions. In some cases, serine (S) residues are used to substitutecysteine residues. Nonlimiting examples of such mutations may includeC4S, C5S and/or C7S. In recombinant GPCs comprising N-terminal prodomainregions from TGF-β1, cysteine residues residing at amino acid positionnumber 4 may be mutated. In recombinant GPCs comprising N-terminalprodomain regions from TGF-β2, cysteine residues residing at amino acidposition number 5 may be mutated In recombinant GPCs comprisingN-terminal prodomain regions from TGF-β3, cysteine residues at position7 may be mutated.

In some cases, one or more cysteine in one or more other region of GPCsmay be substituted or deleted. In some embodiments, such GPCmodifications may promote the release of mature growth factor fromprodomains. In some cases, such cysteines may include those present inone or more of mature growth factors, alpha 2 helices, fasteners,latency lassos and/or bow-tie regions.

In some embodiments, recombinant proteins of the present invention maycomprise protein modules derived from one or more species, includingmammals, including, but not limited to mice, rats, rabbits, pigs,monkeys and/or humans. Recombinant proteins may comprise one or moreamino acids from one or more amino acid sequences derived from one ormore non-human protein sequences listed in Table 8. In some cases,recombinant proteins of the present invention may comprise suchsequences with or without the native signal peptide.

TABLE 8 Non-human proteins SEQ ID Protein Species Sequence NO proTGF-β1Mouse LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 116GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF-β1  CynoLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 117GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF-β1 MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 118 C4S (LAP)GPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMAT PLERAQHLHSSRHRR proTGF-β1 CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 119 C4S (LAP)GPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMAT PLERAQHLQSSRHRR proTGF-β1 MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 120 C4S D2GGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLS NMIVRSCKCS proTGF-β1 MouseLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 121 C4SGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF-β1 CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 122 C4SGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQ LSNMIVRSCKCS proTGF-β1 CynoLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPP 123 C4S D2GGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSKDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHGALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQL SNMIVRSCKCS LRRC32 CynoMSPQILLLLALLTLGLAAQHQDKVACKMVDKKVSCQG 124LGLLQVPLVLPPDTETLDLSGNQLRSILASPLGFYTALRHLDLSTNEINFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPHLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSTPNGNVSARPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRSGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPNEPGPASCVAFSGIASLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALTGLEASLEVLALQGNGLTVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVSAILLTTLATCCCVRRQKFNQQYKA proGDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 125KLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 126 AxxAKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 127 D76AKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS proGDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 128 AxxA D76AKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRC GCS GDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 129 prodomainKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRR GDF-8 MouseNEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILS 130 prodomainKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRADSS D76ADGSLEDDDYHATTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKV TDTPKRSRR proGDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 131LRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 132 AxxALRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 133 D76ALRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS proGDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 134 AxxA D76ALRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS GDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 135 prodomainLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR GDF-8 CynoNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSK 136 prodomainLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRADSSD D76AGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVT DTPKRSRR proGDF-11 MouseAEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 137CVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCSproGDF-11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 138 AxxACVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCSproGDF-11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 139 AxxA D96ACVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTP TKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCSproGDF-11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 140 D96ACVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPT KMSPINMLYFNDKQQIIYGKIPGMVVDRCGCSGDF-11 Mouse AEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 141 prodomainCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR GDF-11 MouseAEGPAAAAAAAAAAAGVGGERSSRPAPSAPPEPDGCPV 142 prodomainCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLL D96APKAPPLQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR LTBP3 CYNOMPGPRGAPGGLAPEMRGAGAAGLLALLLLLGLGGRVE 143GGPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGSNMTLIGENGHSTDTLTGSGFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAGGAGGGTGGSGPGLSRAGALSTGALPPLAPEGDSVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIESSNAEGAAPSQHLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAFKEICPAGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPPPEDTEEERGVTTDSPVSEERSVQQSHPTATTSPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCVDVDECEAGDVCDNGICTNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECTQDPGLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPQAGPALTFDDCCCRQGRGWGAQCRPCPPRGAGSQCPTSQSESNSFWDTSPLLLGKPRRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSE RCVNTSGSFRCVCKAGFARSRPHGACVPQRRRLTBP3 Mouse MPGPRGAAHGLAPAMHQAGALGLLALLLLALLGPGGG 144AEGGPAGERGTGGGGALARERFKVVFAPVICKRTCLKGQCRDSCQQGSNMTLIGENGHSTDTLTGSAFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAAGTGAGTGSSGPGLARTGAMSTGPLPPLAPEGESVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIEGPNAEGPASSQHLLPHPKPPHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPVPVRGEVGADCPQGYKRLNSTHCQDINECAMPGNVCHGDCLNNPGSYRCVCPPGHSLGPLAAQCIADKPEEKSLCFRLVSTEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPADGTAAFKEICPGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSRAPPLEDTEEERGVTMDPPVSEERSVQQSHPTTTTSPPRPYPELISRPSPPTFHRFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGQCVPGPSDYSCHCNAGYRSHPQHRYCVDVNECEAEPCGPGKGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPICEDIDECRDPSTCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECSEGTPCSPGWCENLPGSYRCTCAQYEPAQDGLSCIDVDECEAGKVCQDGICTNTPGSFQCQCLSGYHLSRDRSRCEDIDECDFPAACIGGDCINTNGSYRCLCPLGHRLVGGRKCKKDIDECSQDPGLCLPHACENLQGSYVCVCDEGFTLTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLVPDGKRLHSGQQHCELCIPAHRDIDECILFGAEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQAQCLIPERWSTPQRDVKCAGASEERTACVWGPWAGPALTFDDCCCRQPRLGTQCRPCPPRGTGSQCPTSQSESNSFWDTSPLLLGKSPRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGFTRSRPHGPACLSAAADDAAIAHTSVIDHRGYFH LTBP1 CynoMAGAWLRWGLLLWAGLLASSAHGRLRRITYVVHPGPG 145LAAGALPLSGPPRSRTFNVALNARYSRSSAAAGAPSRASPGVPSERTRRTSKPGGAALQGLRPPPPPPPEPARPAAPGGQLHPKPGGHPAAAPFAKQGRQVVRSKVPQETQSSGGSRLQVHQKQQLQGVNVCGGRCCHGWSKAPGSQRCTKRSCVPPCQNGGMCLRPQLCVCKPGTKGKACETIAAQDTSSPVFGGQSPGAASSWGPPEQAAKHTSSKKADTLPRVSPVAQMTLTLKPKPSVGLPQQIHSQVTPLSSQSVMIHHSQTQEYVLKPKYFPAQKGISGEQSTEGSFPLRYVQDQVAAPFQLSNHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGHAADTLTATNFRVVLCHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPLTVTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPVISEEKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHHHVGKGPVFVKPKNTQPVAKSTHPPPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYKFSEQQRKCVDIDECTQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCTNGDCSNLEGSYMCSCHKGYTRTPDHKHCKDIDECQQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHHHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDVEQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYAPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTEGSYKCLCLPG YVPSDKPNYCTPLNTALNLEKDSDLE LTBP1Smouse NHTGRIKVVFTPSICKVTCTKGNCQNSCQKGNTTTLISE 146NGHAADTLTATNFRVVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVLGASMPKLYQHAQQQGKALGSHVIHSTHTLPLTMTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDSPGGQKVKEAQPGQSQVSYQGLPVQKTQTVHSTYSHQQLIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKQSYHGYTQMMECLQGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCSCKMGFGPDPTFSSCVPDPPVISEEKGPCYRLVSPGRHCMHPLSVHLTKQICCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIHQHIGKEAVYVKPKNTQPVAKSTHPPPLPAKEEPVEALTSSWEHGPRGAEPEVVTAPPEKEIPSLDQEKTRLEPGQPQLSPGVSTIHLHPQFPVVVEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYKFSEQLRKCVDIDECAQVRHLCSQGRCENTEGSFLCVCPAGFMASEEGTNCIDVDECLRPDMCRDGRCINTAGAFRCEYCDSGYRMSRRGYCEDIDECLKPSTCPEEQCVNTPGSYQCVPCTEGFRGWNGQCLDVDECLQPKVCTNGSCTNLEGSYMCSCHRGYSPTPDHRHCQDIDECQQGNLCMNGQCRNTDGSFRCTCGQGYQLSAAKDQCEDIDECEHHHLCSHGQCRNTEGSFQCVCNQGYRASVLGDHCEDINECLEDSSVCQGGDCINTAGSYDCTCPDGFQLNDNKGCQDINECAQPGLCGSHGECLNTQGSFHCVCEQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRVTEDSGVDRQPREEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGAGWGDNCEIFPCPVQGTAEFTEMCPRGKGLVPAGESSYDTGGENYKDADECLLFGEEICKNGYCLNTQPGYECYCKQGTYYDPVKLQCFDMDECQDPNSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCVQPTESNEQIEETDVYQDLCWEHLSEEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPMKDSDDYAQLCNIPVTGRRRPYGRDALVDFSEQYGPETDPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDMAKMTCVDVNECSELNNRMSLCKNAKCINTEGSY KCLCLPGYIPSDKPNYCTPLNSALNLDKESDLEGARP mouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALY 147LSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSLTCISDFSLQQLQVLDLSCNSIEAFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTSLRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQDNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFGSQEELSLSLVRPEDCEKGGLKNVNLILLLSFTLVS AIVLTTLATICFLRRQKLSQQYKA sGARPmouse ISQRREQVPCRTVNKEALCHGLGLLQVPSVLSLDIQALY 148LSGNQLQSILVSPLGFYTALRHLDLSDNQISFLQAGVFQALPYLEHLNLAHNRLATGMALNSGGLGRLPLLVSLDLSGNSLHGNLVERLLGETPRLRTLSLAENSLTRLARHTFWGMPAVEQLDLHSNVLMDIEDGAFEALPHLTHLNLSRNSLTCISDFSLQQLQVLDLSCNSIEAFQTAPEPQAQFQLAWLDLRENKLLHFPDLAVFPRLIYLNVSNNLIQLPAGLPRGSEDLHAPSEGWSASPLSNPSRNASTHPLSQLLNLDLSYNEIELVPASFLEHLTSLRFLNLSRNCLRSFEARQVDSLPCLVLLDLSHNVLEALELGTKVLGSLQTLLLQDNALQELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLHVLNMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALVGLEASLEVLELQGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTRAVSLEVLDLRNNSFSLLPGNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDL ICRFGSQEELSLSLVRPEDCEKGGLKNVNLRRC33 mouse WRSGPGTATAASQGGCKVVDGVADCRGLNLASVPSSLP 149PHSRMLILDANPLKDLWNHSLQAYPRLENLSLHSCHLDRISHYAFREQGHLRNLVLADNRLSENYKESAAALHTLLGLRRLDLSGNSLTEDMAALMLQNLSSLEVVSLARNTLMRLDDSIFEGLEHLVELDLQRNYIFEIEGGAFDGLTELRRLNLAYNNLPCIVDFSLTQLRFLNVSYNILEWFLAAREEVAFELEILDLSHNQLLFFPLLPQCGKLHTLLLQDNNMGFYRELYNTSSPQEMVAQFLLVDGNVTNITTVNLWEEFSSSDLSALRFLDMSQNQFRHLPDGFLKKTPSLSHLNLNQNCLKMLHIREHEPPGALTELDLSHNQLAELHLAPGLTGSLRNLRVFNLSSNQLLGVPTGLFDNASSITTIDMSHNQISLCPQMVPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALQDCPFQGTSLTHLDLSSNWGVLNGSISPLWAVAPTLQVLSLRDVGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLALRTLDLRRNSLTALPQRVVSEQPLRGLQTIYLSQNPYDCCGVEGWGALQQHFKTVADLSMVTCNLSSKIVRVVELPEGLPQGCKWEQVDTGLFYLVLILPSCLTLLVACTVVFLTF KKPLLQVIKSRCHWSSIY sLRRC33 mouseWRSGPGTATAASQGGCKVVDGVADCRGLNLASVPSSLP 150PHSRMLILDANPLKDLWNHSLQAYPRLENLSLHSCHLDRISHYAFREQGHLRNLVLADNRLSENYKESAAALHTLLGLRRLDLSGNSLTEDMAALMLQNLSSLEVVSLARNTLMRLDDSIFEGLEHLVELDLQRNYIFEIEGGAFDGLTELRRLNLAYNNLPCIVDFSLTQLRFLNVSYNILEWFLAAREEVAFELEILDLSHNQLLFFPLLPQCGKLHTLLLQDNNMGFYRELYNTSSPQEMVAQFLLVDGNVTNITTVNLWEEFSSSDLSALRFLDMSQNQFRHLPDGFLKKTPSLSHLNLNQNCLKMLHIREHEPPGALTELDLSHNQLAELHLAPGLTGSLRNLRVFNLSSNQLLGVPTGLFDNASSITTIDMSHNQISLCPQMVPVDWEGPPSCVDFRNMGSLRSLSLDGCGLKALQDCPFQGTSLTHLDLSSNWGVLNGSISPLWAVAPTLQVLSLRDVGLGSGAAEMDFSAFGNLRALDLSGNSLTSFPKFKGSLALRTLDLRRNSLTALPQRVVSEQPLRGLQTIYLSQNPYDCCGVEGWGALQQHFKTVADLSMVTCNLSSKIVRVVELPE GLPQGCKWEQVDTGL LRRC33 CynoWRDRSVTATAASQRGCKLVGGDTDCRGQSLASVPSSLP 151PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERIGRGAFQEQGHLRSLVLGDNCLSENYKETAAALHTLPGLQTLDLSGNSLTEDMAALMLQNLSSLQSVSLARNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLTELRHLNLAYNNLPCIVDFGLTQLRSLNVSYNVLEWFLAAGGEAAFELETLDLSHNQLLFFPLLPQYSKLHTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVNLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLNQNCLMTLHIREHEPPGALTELDLSHNQLSELHLTPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGLHSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQQGQTVADWATVTCNLSSKIIRLAELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLVACTLIVLTFKKPLL QVIKSRCHWSSVY sLRRC33 CynoWRDRSVTATAASQRGCKLVGGDTDCRGQSLASVPSSLP 152PHARTLILDANPLKALWNHSLQPYPLLESLSLHSCHLERIGRGAFQEQGHLRSLVLGDNCLSENYKETAAALHTLPGLQTLDLSGNSLTEDMAALMLQNLSSLQSVSLARNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLTELRHLNLAYNNLPCIVDFGLTQLRSLNVSYNVLEWFLAAGGEAAFELETLDLSHNQLLFFPLLPQYSKLHTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVNLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLNQNCLMTLHIREHEPPGALTELDLSHNQLSELHLTPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTSLDLSSNWGVLNGSLAPLRDVAPMLQVLSLRNMGLHSNFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQQGQTVADWATVTCNLSSKIIRLAELPGGVPR DCKWERLDLGL

In some embodiments, recombinant proteins may be combined and/orcomplexed with one or more additional recombinant components. Suchcomponents may include extracellular proteins known to associate withGPCs including, but not limited to LTBPs, fibrillins, perlecan, GASP1/2proteins, follistatin, follistatin-related gene (FLRG), decorin and/orGARP (including, but not limited to recombinant forms of such proteins).Some recombinant GPCs of the present invention must be co-expressed withone or more of such extracellular proteins for proper expression and/orfolding.

In some embodiments, complexed LTBPs may include, but are not limited toLTBP1, LTBP2, LTBP3 and/or LTBP4. Complexed LTBPs may comprise LTBPfragments and/or mutations. Some recombinant forms of LTBPs complexedwith recombinant GPCs may comprise alternatively spliced variants ofLTBPs. Some such variants of LTBP1 are shortened at the N-terminus,referred to herein as LTBP1S. Some recombinant proteins of the presentinvention may comprise LTBPs, fragments or mutants thereof comprisingthe amino acid sequences listed in Table 9.

TABLE 9 LTBP sequences SEQ ID Protein Sequence NO LTBP1 1265-1443NECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQC 153RSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGY ECYCKQGTYYDPVKLQCFLTBP1 1265-1698 NECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQC 154RSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPLKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDSDLE LTBP1 809-1698PSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPV 155EVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYRFSEQQRKCVDIDECTQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCANGDCSNLEGSYMCSCHKGYTRTPDHKHCRDIDECQQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPLKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLC LPGYVPSDKPNYCTPLNTALNLEKDSDLELTBP1S NHTGRIKVVFTPSICKVTCTKGSCQNSCEKGNTTTLISENGH 156AADTLTATNFRVVICHLPCMNGGQCSSRDKCQCPPNFTGKLCQIPVHGASVPKLYQHSQQPGKALGTHVIHSTHTLPLTVTSQQGVKVKFPPNIVNIHVKHPPEASVQIHQVSRIDGPTGQKTKEAQPGQSQVSYQGLPVQKTQTIHSTYSHQQVIPHVYPVAAKTQLGRCFQETIGSQCGKALPGLSKQEDCCGTVGTSWGFNKCQKCPKKPSYHGYNQMMECLPGYKRVNNTFCQDINECQLQGVCPNGECLNTMGSYRCTCKIGFGPDPTFSSCVPDPPVISEEKGPCYRLVSSGRQCMHPLSVHLTKQLCCCSVGKAWGPHCEKCPLPGTAAFKEICPGGMGYTVSGVHRRRPIFIHHVGKGPVFVKPKNTQPVAKSTHPPPLPAKEEPVEALTFSREHGPGVAEPEVATAPPEKEIPSLDQEKTKLEPGQPQLSPGISTIHLHPQFPVVIEKTSPPVPVEVAPEASTSSASQVIAPTQVTEINECTVNPDICGAGHCINLPVRYTCICYEGYRFSEQQRKCVDIDECTQVQHLCSQGRCENTEGSFLCICPAGFMASEEGTNCIDVDECLRPDVCGEGHCVNTVGAFRCEYCDSGYRMTQRGRCEDIDECLNPSTCPDEQCVNSPGSYQCVPCTEGFRGWNGQCLDVDECLEPNVCANGDCSNLEGSYMCSCHKGYTRTPDHKHCRDIDECQQGNLCVNGQCKNTEGSFRCTCGQGYQLSAAKDQCEDIDECQHRHLCAHGQCRNTEGSFQCVCDQGYRASGLGDHCEDINECLEDKSVCQRGDCINTAGSYDCTCPDGFQLDDNKTCQDINECEHPGLCGPQGECLNTEGSFHCVCQQGFSISADGRTCEDIDECVNNTVCDSHGFCDNTAGSFRCLCYQGFQAPQDGQGCVDVNECELLSGVCGEAFCENVEGSFLCVCADENQEYSPMTGQCRSRTSTDLDVDVDQPKEEKKECYYNLNDASLCDNVLAPNVTKQECCCTSGVGWGDNCEIFPCPVLGTAEFTEMCPKGKGFVPAGESSSEAGGENYKDADECLLFGQEICKNGFCLNTRPGYECYCKQGTYYDPVKLQCFDMDECQDPSSCIDGQCVNTEGSYNCFCTHPMVLDASEKRCIRPAESNEQIEETDVYQDLCWEHLSDEYVCSRPLVGKQTTYTECCCLYGEAWGMQCALCPLKDSDDYAQLCNIPVTGRRQPYGRDALVDFSEQYTPEADPYFIQDRFLNSFEELQAEECGILNGCENGRCVRVQEGYTCDCFDGYHLDTAKMTCVDVNECDELNNRMSLCKNAKCINTDGSYKCLCLPGYVPSDKPNYCTPLNTALNLEKDS DLE LTBP3GPAGERGAGGGGALARERFKVVFAPVICKRTCLKGQCRDS 157CQQGSNMTLIGENGHSTDTLTGSGFRVVVCPLPCMNGGQCSSRNQCLCPPDFTGRFCQVPAGGAGGGTGGSGPGLSRTGALSTGALPPLAPEGDSVASKHAIYAVQVIADPPGPGEGPPAQHAAFLVPLGPGQISAEVQAPPPVVNVRVHHPPEASVQVHRIESSNAESAAPSQHLLPHPKPSHPRPPTQKPLGRCFQDTLPKQPCGSNPLPGLTKQEDCCGSIGTAWGQSKCHKCPQLQYTGVQKPGPVRGEVGADCPQGYKRLNSTHCQDINECAMPGVCRHGDCLNNPGSYRCVCPPGHSLGPSRTQCIADKPEEKSLCFRLVSPEHQCQHPLTTRLTRQLCCCSVGKAWGARCQRCPTDGTAAFKEICPAGKGYHILTSHQTLTIQGESDFSLFLHPDGPPKPQQLPESPSQAPPPEDTEEERGVTTDSPVSEERSVQQSHPTATTTPARPYPELISRPSPPTMRWFLPDLPPSRSAVEIAPTQVTETDECRLNQNICGHGECVPGPPDYSCHCNPGYRSHPQHRYCVDVNECEAEPCGPGRGICMNTGGSYNCHCNRGYRLHVGAGGRSCVDLNECAKPHLCGDGGFCINFPGHYKCNCYPGYRLKASRPPVCEDIDECRDPSSCPDGKCENKPGSFKCIACQPGYRSQGGGACRDVNECAEGSPCSPGWCENLPGSFRCTCAQGYAPAPDGRSCLDVDECEAGDVCDNGICSNTPGSFQCQCLSGYHLSRDRSHCEDIDECDFPAACIGGDCINTNGSYRCLCPQGHRLVGGRKCQDIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDCCCRQGRGWGAQCRPCPPRGAGSHCPTSQSESNSFWDTSPLLLGKPPRDEDSSEEDSDECRCVSGRCVPRPGGAVCECPGGFQLDASRARCVDIDECRELNQRGLLCKSERCVNTSGSFRCVCKAGFARSRPHGA CVPQRRR

In some embodiments, LTBPs may comprise detectable labels. Detectablelabels may be used to allow for detection and/or isolation ofrecombinant proteins comprising LTBPs. Some detectable labels maycomprise biotin labels, polyhistidine tags and/or flag tags. Such tagsmay be used to isolate tagged proteins. Proteins produced may compriseadditional amino acids encoding one or more 3C protease cleavage site.Such sites allow for cleavage at the 3C protease cleavage site upontreatment with 3C protease, including, but not limited to rhinovirus 3Cprotease. Such cleavage sites may be introduced to allow for removal ofdetectable labels from recombinant proteins.

In some embodiments, GARPs, including, but not limited to recombinantforms of GARP, may be complexed with recombinant GPCs. Some recombinantGPCs of the present invention may be co-expressed with GARPs to ensureproper folding and/or expression. In other embodiments, the GARPhomologue, leucine rich repeat containing 33 (LRRC33) or fragmentsand/or mutants thereof may be substituted for GARP [also referred toherein as leucine rich repeat containing 32 (LRRC32.)] Such LRRC33fragments and/or mutants may comprise one or more regions from theLRRC33 sequence listed in Table 10 below. Recombinant GARPs may alsocomprise mutants and/or GARP fragments. Some recombinant GARPs may besoluble (referred to herein as sGARP).

In some embodiments, recombinant GARPs may comprise one or more aminoacid sequences listed in Table 10. Some recombinant GARPs used hereinmay be expressed without the N-terminal residues AQ. Expressed GARPs maycomprise detectable labels. Such detectable labels may be used to allowfor detection and/or isolation. Some detectable labels may comprisebiotin labels, polyhistidine tags and/or flag tags. Such tags may beused to isolate tagged proteins. Proteins produced may compriseadditional amino acids encoding one or more 3C protease cleavage site.Such sites allow for cleavage at the 3C protease cleavage site upontreatment with 3C protease, including, but not limited to rhinovirus 3Cprotease. 3C protease cleavage sites may be introduced to allow forremoval of detectable labels from recombinant proteins.

TABLE 10 GARP sequences SEQ ID Protein Sequence NO GARPAQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLS 158GNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVS AILLTTLAACCCVRRQKFNQQYKAsGARP AQHQDKVPCKMVDKKVSCQVLGLLQVPSVLPPDTETLDLS 159GNQLRSILASPLGFYTALRHLDLSTNEISFLQPGAFQALTHLEHLSLAHNRLAMATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTFRDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSCNSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQDSKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNLSRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPYTFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGAFLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNLAENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNIN LRRC33WRNRSGTATAASQGVCKLVGGAADCRGQSLASVPSSLPPH 160ARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKIIRVTELPGGVPRDCKWERLDLGLLYLVLILPSCLTLLVAC TVIVLTFKKPLLQVIKSRCHWSSVYsLRRC33 WRNRSGTATAASQGVCKLVGGAADCRGQSLASVPSSLPPH 161ARMLTLDANPLKTLWNHSLQPYPLLESLSLHSCHLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALMLQNLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLTRLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPREMVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIREHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCPLPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQVLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGLRTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSS KIIRVTELPGGVPRDCKWERLDLGL

GPCs bound to LTBPs may adopt three dimensional conformations that aredistinct from conformations found with GPCs bound to GARP or othermatrix proteins. This may be due, in some cases, to the presence ofcysteines available on LTBP for disulfide bond formation with GPCs thatcomprise a different distance from one another than correspondingcysteines available for disulfide bond formation on GARP. Suchdifferences in three dimensional conformations may provide uniqueconformation-dependent epitopes on GPCs. In some embodiments, antibodiesof the invention are directed to such conformation-dependent epitopes.Such antibodies may function selectively to activate or inhibit growthfactor activity depending on the identity of bound protein (e.g. LTBP orGARP.) In some cases, different conformation-dependent epitopes may bepresent on N-terminal alpha helices of proTGF-β when bound to LTBP orGARP.

Recombinant proteins of the present invention may be coexpressed withGDF-associated serum protein (GASP) 1 and/or GASP-2. Such recombinantproteins may include, but are not limited to GDF-8 and/or GDF-11. GASPsare circulating proteins that bind and prevent activity of GDF-8 andGDF-11 (Hill, J. J. et al., 2003. Mol Endocrinology. 17(6):1144-54 andHill, J. J. et al., 2002. JBC. 277(43):40735-41, the contents of each ofwhich are herein incorporated by reference in their entirety.)Interestingly, GDF-8 and GDF-11 growth factors are not found free inserum. About 70% are in GPCs with the remaining 30% associated withGASPs as well as other proteins (e.g. follistatin, follistatin-likerelated gene and decorin.) Studies using mice lacking expression ofGASP-1 and/or GASP-2 display phenotypes indicative of myostatin and/orGDF-11 overactivity (Lee et al., 2013. PNAS. 110(39):E3713-22.) GASPbound GDF-8 and/or GDF-11 are unable to bind type II receptors andtransmit related cellular signals.

Some recombinant proteins may be coexpressed with perlecan. Suchrecombinant proteins may include, but are not limited to GDF-8. Studiesby Sengle et al (Sengle et al., 2011. J Biol Chem. 286(7):5087-99, thecontents of which are herein incorporated by reference in theirentirety) found that the GDF-8 prodomain associates with perlecan.Further studies indicate that perlecan knockout leads to muscularhypertrophy, suggesting that the interaction between GDF-8 and perlecanmay contribute to GDF-8 activity (Xu et al. 2010. Matrix Biol.29(6):461-70.)

In some cases, recombinant proteins of the invention may be coexpressedwith follistatin and/or FLRG. Such recombinant proteins may include, butare not limited to GDF-8. Both follistatin and FLRG are known toantagonize some TGF-β family member proteins, including, but not limitedto GDF-8 (Lee, S-J. et al., 2010. Mol Endocrinol. 24(10):1998-2008,Takehara-Kasamatsu, Y. et al., 2007. J Med Invest. 54(3-4):276-88, thecontents of each of which are herein incorporated by reference in theirentirety.) Follistatin has been shown to block GDF-8 activity by bindingto the free growth factor and preventing receptor binding. Bothfollistatin and FLRG are implicated in modulating growth factor activityduring development.

In some embodiments, recombinant proteins of the invention may becoexpressed with decorin. Such recombinant proteins may include, but arenot limited to TGF-β and GDF-8. Decorin is a known antagonist of TGF-βactivity (Zhu, J. et al., 2007. J Biol Chem. 282:25852-63, the cotentsof which are herein incorporated by reference in their entirety) and mayalso antagonize other TGF-β family members, including, but not limitedto GDF-8. Decorin-dependent inhibition of TGF-β and GDF-8 activity hasbeen shown to reduce fibrosis in various tissues. Decorin expression hasalso been shown to increase the expression of follistatin, a knowninhibitor of free GDF-8.

In some embodiments, recombinant proteins of the present invention maycomprise those depicted in FIG. 7. Some recombinant proteins of thepresent invention may comprise one or more features and/or combinationsof protein modules from the embodiments depicted in FIG. 7.

Recombinant Growth Differentiation Factors (GDFs) Activins and Inhibins

Growth differentiation factors (GDFs), activins and inhibins are TGF-βfamily member proteins involved in a number of cellular and/ordevelopmental activities. In some embodiments of the present invention,recombinant proteins may comprise one or more protein modules from oneor more GDFs, activins and/or inhibins. In further embodiments, GDFprotein modules may comprise GDF-8 and/or GDF-11 protein modules.

GDF-8 and GDF-11, which are secreted as latent complexes (Sengle et al.,2011. J Biol Chem. 286(7):5087-99; Ge et al., 2005. Mol Cel Biol.25(14):5846-58) show conservation of the fastener residues (Lys 27 andTyr 75 of TGF-β1; see FIGS. 8A-8G.) GDF-8 (also referred to herein asmyostatin) is involved in regulating muscle mass, and its deficiencyincreases muscle mass in multiple species, including humans(Rodino-Klapac, L. R. et al., 2009. Muscle Nerve. 39(3):283-96). GDF-8may be found in the circulation in latent form, but may also be storedin the extracellular matrix, bound to LTBP3 (Anderson et al., 2007. JBiol Chem. 283(11):7027-35) or perlecan (Sengle et al., 2011. J BiolChem. 286(7):5087-99.) While complexed with its prodomain, GDF-8 isunable to participate in receptor binding with the type II receptor,ActRIIB (Sengle et al., 2008. J Mol Biol. 381(4):1025-39.) While GDF-8is expressed primarily in muscle, GDF-11 expression is more systemic andits activity is thought to be involved in multiple processes (Lee etal., 2013. PNAS. 110(39):E3713-22.). It is believed to be involved indevelopment of multiple tissues, including, but not limited to theretina, kidney, pancreas and olfactory system. It is also believed to bea circulating factor in the blood (Sinha, M. et al., 2014. ScienceExpress. 10.1126/science.1251152, p 2-6 and Katsimpardi, L. et al.,2014. Science Express. 10.1126/science.1251141, the contents of each ofwhich are herein incorporated by reference in their entirety.)

GDF-8 and GDF-11 also share considerable homology. While the prodomainsonly share 48% homology, GDF-8 and GDF-11 growth factor domains share90% homology (60% homology when prodomains and growth factors are takentogether.)

Release of GDF-8 and GDF-11 from latent GPCs requires cleavage of theprodomains at the BMP/tolloid cleavage site (located between Arg 75 andAsp 76 in GDF-8 and between Gly 97 and Asp 98 in GDF-11) by BMP1/tolloidmetalloproteinases. This cleavage is between the α2 helix and thefastener. Thus at least two different methods of unfastening thestraitjacket, force and proteolysis, can release family members fromlatency.

In some embodiments, recombinant proteins of the present inventioncomprising GDFs may comprise sequences listed in Table 11 or fragmentsthereof.

TABLE 11 Recombinant GDFs SEQ ID Protein Sequence NO proGDF-8NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE   5TAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVV DRCGCS GDF-8 prodomainNENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE  70TAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTF PGPGEDGLNPFLEVKVTDTPKRSRRGDF-8 prodomain NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE 162 D76ATAPNISKDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTF PGPGEDGLNPFLEVKVTDTPKRSRRproGDF-8 AXXA NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE 163TAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVV DRCGCS proGDF-8 D76ANENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE 164TAPNISKDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVV DRCGCS proGDF-8 AXXANENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLE 165 D76ATAPNISKDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKASRADFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVV DRCGCS proGDF-11AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC   4VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF-11 D98AAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 166VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF-11 D2GAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 167VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSGNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF-11 AxxAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 168VWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS proGDF-11 AxxAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 169 D98AVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKASRANLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-11AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC 170 prodomain D98AVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGAALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR GDF-11AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVC  71 prodomainVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGL HPFMELRVLENTKRSRR

Activins and inhibins are TGF-β family member proteins, the activity ofeach of which often results in opposing functions (Bilezikjian et al2012.) Like other family members, these proteins occur physiologicallyas dimers. Activins and inhibins are constructed in part from the sameβ-subunits, that may include inhibin-beta A, inhibin-beta B,inhibin-beta C and inhibin-beta E (referred to herein as β-subunit A, B,C and E, respectively.) The difference between activins and inhibins,structurally, is that activins are β-subunit dimers while inhibins areheterodimers, wherein the second subunit is inhibin-α. Activins arenamed for their subunit pairs, such that activin A comprises a homodimerof two A subunits, activin AB comprises a dimer of A and B subunits, Bcomprises a dimer of B subunits, etc. (Muenster et al 2011.) Activinsare involved in a variety of functions that may include, but are notlimited to cell growth, differentiation, programmed cell death,endocrine functions, cellular metabolism, bone growth, etc. They areespecially recognized for their control of reproductive hormone cycles.Activin and inhibin signaling often functions antagonistically in thisregard.

In some embodiments, recombinant proteins of the present invention maycomprise integrins. Integrins are cell surface heterodimers formed byalpha and beta subunits, each of which has a transmembrane domain and inthe N-terminal portion of the extracellular domain come together to formthe ligand binding site. Recombinant proteins of the present inventionmay comprise integrins and/or integrin subunits. Such integrins and/orintegrin subunits may comprise any of those disclosed in U.S.Provisional Patent Application No. 61/722,919 filed Nov. 6, 2012, thecontents of which are herein incorporated by reference in theirentirety.

Recombinant proteins of the invention may include intercellular adhesionmolecule 1 (ICAM-1). In some cases, ICAM-1 proteins of the presentinvention may be used as control proteins during antibody developmentand/or antibody testing. In some cases, ICAM-1 may be used as a controlduring selection of binding molecules using phage display technologies.In some cases, ICAM-1 proteins of the invention comprise one or moredetectable label. Detectable labels may include, for example, histidinetags.

Chimeric Proteins

In some embodiments, recombinant proteins of the present invention maycomprise chimeric proteins. As used herein, the term “chimeric protein”refers to a protein comprising one or more protein modules from at leasttwo different proteins [formed from the same gene (e.g. variants arisingfrom alternative splicing) or from different genes]. Chimeric proteinsmay comprise protein modules from two or more TGF-β family memberproteins. Such chimeric proteins may comprise protein modules fromTGF-β1, TGF-β2 and/or TGF-β3. Some chimeric proteins of the presentinvention may comprise protein modules including, but not limited to theprotein modules and/or amino acid sequences listed in Table 12 (residuenumbers correspond to the pro-protein sequences listed in Table 1.) Somechimeric proteins of the present invention may comprise protein modulescomprising amino acid sequences similar to those in Table 12, butcomprising additional or fewer amino acids than those listed. Suchmodules may comprise about 1 more or fewer amino acids, about 2 more orfewer amino acids, about 3 more or fewer amino acids, about 4 more orfewer amino acids, about 5 more or fewer amino acids, about 6 more orfewer amino acids, about 7 more or fewer amino acids, about 8 more orfewer amino acids, about 9 more or fewer amino acids, about 10 more orfewer amino acids or greater than 10 more or fewer amino acids onN-terminal and/or C-terminal ends.

TABLE 12 Protein modules SEQ ID Protein Residues Sequence NO TGF-β1  1-74 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV 171PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADY TGF-β1   1-207LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV 172PPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIE GFRLSAHCSCDSRDNTLQVDI TGF-β1 46-end EAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTR 173VLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF-β1 47-end AVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRV 174LMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYY VGRKPKVEQLSNMIVRSCKCS TGF-β1 74-249 YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSE 175LREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATI HGMNRPFLLLMATPLERAQHLQSSRHRRTGF-β1  74-end YYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSE 176LREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVP QALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSTGF-β1  75-249 YAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSEL 177REAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIH GMNRPFLLLMATPLERAQHLQSSRHRR TGF-β1 75-end YAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSEL 178REAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA LEPLPIVYYVGRKPKVEQLSNMIVRSCKCSTGF-β1 228-361 FLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNC 179CVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPI VYYVGRKPKVEQLSNMIVRSCKCS TGF-β1250-361 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP  44KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSC KCS TGF-β2 232-260FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP  65 TGF-β2 236-254 GTSTYTSGDQKTIKSTRKK180 TGF-β3   1-46 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEP  43 TVMTHVPTGF-β3   1-79 SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEP 181TVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQEN TESE TGF-β3  80-280YYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNV 182SSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDEIHNPHLILMMIPP HRLDNPGQGGQRKKR TGF-β3 281-392ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEP  46KGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCK CS GDF-8   1-75NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQIL 183SKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQR GDF-8   1-64NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQIL  72 SKLRLETAPNISKDVIRQLLPKAPPLGDF-8  75-end RDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGK 184PKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGK EQIIYGKIPAMVVDRCGCS GDF8  65-endRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTES 185DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSP INMLYFNGKEQIIYGKIPAMVVDRCGCS GDF8 65-243 RELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTES  77DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHD LAVTFPGPGEDGLNPFLEVKVTDTPKRSRRGDF-8  76-243 DDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKP 186KCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGED GLNPFLEVKVTDTPKRSRR GDF-8 244-352DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKR  74YKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF-11   1-86AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD  73GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISRE VVKQLLPKAPPL GDF-11   1-96AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 187GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISRE VVKQLLPKAPPLQQILDLHDFQ GDF-11  1-108 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPD 188GCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEE GDF-11  97-274GDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDG 189SPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTD LAVTSLGPGAEGLHPFMELRVLENTKRSRRGDF-11  87-274 QQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQ  78ETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRS RR GDF-11 275-383NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKR  75YKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS Inhibin   1-64SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 190 Beta AVKKHILNMLHLKKRPDVTQPVPKAALL Inhibin   1-76SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEA 191 Beta AVKKHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVG KVG Inhibin  65-288NAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQT 192 Beta ASEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RR Inhibin  65-289NAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQT 193 Beta ASEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRR Inhibin  65-290NAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQT 194 Beta ASEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRR Inhibin  77-289ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 195 Beta ATLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQARQSEDHPHRRRR Inhibin 77-290 ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 196 Beta ATLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQARQSEDHPHRRRRRInhibin  77-end ENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARK 197 Beta ATLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGA DEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS Inhibin 291-406GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHA 198 Beta ANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEE CGCS

In some embodiments, chimeric proteins of the present invention maycomprise combinations of any of the protein modules listed in Table 12.Some chimeric proteins comprising GPCs may comprise protein modules thathave been substituted with any of the protein modules listed in Table12.

In some embodiments, chimeric proteins may comprise protein modules fromGDFs and/or inhibins. Such GDFs may include GDF-11 and/or GDF-8. Somesuch chimeric proteins may comprise a prodomain from GDF-11 and a growthfactor from GDF-8. In such embodiments, chimeric proteins may comprisesubstituted N-terminal regions between GDF-11 and GDF-8. In otherembodiments, chimeric proteins may comprise a prodomain from GDF-8 and agrowth factor from GDF-11. Such chimeric proteins may comprise aminoacid residues 1-108 from GDF-11 and amino acid residues 90-the end ofthe protein from GDF-8. Some chimeric proteins may comprise an armregion from GDF-11.

Some chimerics of the present invention may comprise GDF-8 comprising anarm region of GDF-11. Such chimerics may be unstable due to steric clashbetween residue F95 from the GDF-11 arm and the α2 helix of the chimericGPC. Therefore, in some cases, GDF8/GDF11/Activin chimeras may bedesigned so that the ARM region of such chimeric proteins contains theα2 helix. Furthermore, F95 may be an important residue in conferringlatency for GDF11. This residue is in a similar position as aCamurati-Engelmann mutation found in TGF-β1, Y81H (see FIGS. 8A-8G),thus, mutation of this residue to a smaller amino acid, such as anAlanine, may be carried out to promote dissociation of the mature GDF11growth factor from the GPC. Such mutants may be useful as positivecontrol molecules in designing assays to screen for GDF11 activatingantibodies.

In some embodiments, chimeric proteins of the present invention maycomprise protein module combinations including, but not limited to thecombinations of protein modules and/or amino acid sequences listed inTable 13. Some chimeric proteins of the present invention may compriseprotein modules comprising amino acid sequences similar to those inTable 13, but comprising additional or fewer amino acids than thoselisted. Such amino acid sequences may comprise about 1 more or feweramino acids, about 2 more or fewer amino acids, about 3 more or feweramino acids, about 4 more or fewer amino acids, about 5 more or feweramino acids, about 6 more or fewer amino acids, about 7 more or feweramino acids, about 8 more or fewer amino acids, about 9 more or feweramino acids, about 10 more or fewer amino acids or greater than 10 moreor fewer amino acids on N-terminal and/or C-terminal ends.

TABLE 13 Protein module combinations Protein Protein Protein SEQ modulemodule module ID 1 2 3 Chimeric Sequence NO TGF-β2 TGF-β1 N/ASLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 199 LAP growthDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACE factorRERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPY FRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAE GEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQ KTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCS TGF-β3 TGF-β1 N/ASLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 200 LAP growthPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEG factorCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCP KGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRG TAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDL GRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVE QLSNMIVRSCKCS TGF-β3 TGF-β1 N/ASLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 201 (1-46) (47-end)PEPTVMTHVPAVLALYNSTRDRVAGESAEPEPEP EADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHV ELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDI NGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRK DLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVG RKPKVEQLSNMIVRSCKCS TGF-β3 TGF-β1 N/ASLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 202 (1-79) (75-end)PEPTVMTHVPYQVLALYNSTRELLEEMHGEREEG CTQENTESEYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLK VEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNT LQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLY IDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS TGF-β1 TGF-β3TGF-β1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 203 (1-74) (80-280) (250-GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE 361)ADYYYAKEIHKFDMIQGLAEHNELAVCPKGITSK VFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWL SFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKK QKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEP KGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNM IVRSCKCS TGF-β3 TGF-β1 TGF-β3SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSP 204 (1-79) (75-249) (281-PEPTVMTHVPYQVLALYNSTRELLEEMHGEREEG 392)CTQENTESEYAKEVTRVLMVETHNEIYDKFKQST HSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPL ERAQHLQSSRHRRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSA DTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS TGF-β1 TGF-β2 TGF-β1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 205 (1-207) trigger (228-GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop 361)ADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF ShortFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVE (236-LYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV 254)VRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDIG TSTYTSGDQKTIKSTRKKFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLG WKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS TGF-β1 TGF-β2 TGF-β1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQ 206 (1-207) trigger (228-GEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPE loop 361)ADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF LongFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVE (232-LYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV 260)VRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDIA GIDGTSTYTSGDQKTIKSTRKKNSGKTPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQ LYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLP IVYYVGRKPKVEQLSNMIVRSCKCS GDF-11GDF-8 GDF-11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 207 (1-96) (76-243) (275-EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA 383)PNISREVVKQLLPKAPPLQQILDLHDFQDDSSDGS LEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILR LIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGP GEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQ CEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-11 GDF-8 GDF-11AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 208 (1-86) (65-243) (275-EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA 383)PNISREVVKQLLPKAPPLRELIDQYDVQRDDSSDG SLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFP GPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS GQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-11 GDF-8 N/AAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 209 (1-96) (76-243)EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA PNISREVVKQLLPKAPPLQQILDLHDFQDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFF KFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKT VLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRR GDF-11 GDF-8 NA AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP210 (1-86) (65-243) EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLRELIDQYDVQRDDSSDG SLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQIL RLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFP GPGEDGLNPFLEVKVTDTPKRSRR GDF-11Inhibin GDF-11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 211 (1-96) Beta A (275-EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA (77-290) 383)PNISREVVKQLLPKAPPLQQILDLHDFQENGYVEI EDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRI ACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRR RRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQAN PRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-11 Inhibin GDF-11 AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 212(1-86) Beta A (275- EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA (65-290) 383)PNISREVVKQLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLD VRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQ ANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS GDF-11 Inhibin N/A AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 213(1-96) Beta A EPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA (77-290)PNISREVVKQLLPKAPPLQQILDLHDFQENGYVEI EDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRI ACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRR RR GDF-11 Inhibin NAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAP 214 (1-86) Beta AEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEA (65-290)PNISREVVKQLLPKAPPLNAIRKLHVGKVGENGY VEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTK VTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLD VRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHR RRRR GDF-8 GDF-11 GDF-8NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 215 (1-75) (97-274) (244-QILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQY 352)DVQRGDALQPEDFLEEDEYHATTETVISMAQETD PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTH LVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF-8 GDF-11 GDF-8 NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI216 (1-64) (87-274) (244- QILSKLRLETAPNISKDVIRQLLPKAPPLQQILDLH 352)DFQGDALQPEDFLEEDEYHATTETVISMAQETDP AVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF-8 GDF-11 N/A NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 217(1-75) (97-274) QILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRGDALQPEDFLEEDEYHATTETVISMAQETD PAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRR HIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELR VLENTKRSRR GDF-8 GDF-11 GDF-8NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 218 (1-64) (87-274) (244-QILSKLRLETAPNISKDVIRQLLPKAPPLQQILDLH 352)DFQGDALQPEDFLEEDEYHATTETVISMAQETDP AVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHI RIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRV LENTKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHL VHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF-8 Inhibin GDF-8 NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI219 (1-75) Beta A (244- QILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQY (77-289)352) DVQRENGYVEIEDDIGRRAEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVP KANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLL DQGKSSLDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQAR QSEDHPHRRRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPH THLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS GDF-8 Inhibin GDF-8NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 220 (1-64) Beta A (244-QILSKLRLETAPNISKDVIRQLLPKAPPLNAIRKLH (65-290) 352)VGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITF AESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRDFGLDCDEHSTESRCCRYPL TVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFN GKEQIIYGKIPAMVVDRCGCS GDF-8 InhibinN/A NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 221 (1-75) Beta AQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQY (77-290)DVQRENGYVEIEDDIGRRAEMNELMEQTSEIITFA ESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVG LKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKKEE EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRR GDF-8 Inhibin NA NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKI 222(1-64) Beta A QILSKLRLETAPNISKDVIRQLLPKAPPLNAIRKLH (65-290)VGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITF AESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEE VGLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKKKKK EEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRR Inhibin GDF-8 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM223 Beta A (76-243) Beta A VEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIR (1-76)(291- KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES 406)DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIY LRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAP SGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNII KKDIQNMIVEECGCS Inhibin GDF-8 InhibinSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 224 Beta A (65-243) Beta AVEAVKKHILNMLHLKKRPDVTQPVPKAALLRELI (1-64) (291-DQYDVQRDDSSDGSLEDDDYHATTETIITMPTES 406)DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIY LRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKA LDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAP SGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNII KKDIQNMIVEECGCS Inhibin GDF-8 N/ASPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 225 Beta A (76-243)VEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIR (1-76)KLHVGKVGDDSSDGSLEDDDYHATTETIITMPTES DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR Inhibin GDF-8 NASPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 226 Beta A (65-243)VEAVKKHILNMLHLKKRPDVTQPVPKAALLRELI (1-64)DQYDVQRDDSSDGSLEDDDYHATTETIITMPTES DFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLD MNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKR SRR Inhibin GDF-11 InhibinSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 227 Beta A (97-274) Beta AVEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIR (1-76) (291-KLHVGKVGGDALQPEDFLEEDEYHATTETVISMA 406)QETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQL WVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFR QPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRGLECDGKVNICCKKQFFVSF KDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGQNIIKKDIQNMIVEECGCS InhibinGDF-11 Inhibin SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 228 Beta A (87-274)Beta A VEAVKKHILNMLHLKKRPDVTQPVPKAALLQQIL (1-64) (291-DLHDFQGDALQPEDFLEEDEYHATTETVISMAQE 406)TDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLW VYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQ PQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRRGLECDGKVNICCKKQFFVSFK DIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMS MLYYDDGQNIIKKDIQNMIVEECGCS InhibinGDF-11 N/A SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 229 Beta A (97-274)VEAVKKHILNMLHLKKRPDVTQPVPKAALLNAIR (1-76)KLHVGKVGGDALQPEDFLEEDEYHATTETVISMA QETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGG GRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPF MELRVLENTKRSRR Inhibin GDF-11 NASPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEM 230 Beta A (87-274)VEAVKKHILNMLHLKKRPDVTQPVPKAALLQQIL (1-64)DLHDFQGDALQPEDFLEEDEYHATTETVISMAQE TDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGG RRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFM ELRVLENTKRSRR

Chimeric proteins may be used to characterize and/or map epitopesassociated with GPCs. As used herein, the terms “map” or “mapping” referto the identification, characterization and/or determination of one ormore functional regions of one or more proteins. Such characterizationsmay be necessary for determining interactions between one or moreprotein modules and another agent (e.g. another protein and/or proteinmodule.) Some chimeric proteins may be used to characterize functionsassociated with one or more proteins and/or protein modules.

In some embodiments, chimeric proteins of the present invention maycomprise the sequences listed in Table 14 or fragments thereof

TABLE 14 Chimeric proteins SEQ ID Protein Sequence NO proTGF- LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 231 β1arm3PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE C4SIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIV RSCKCS proTGF- LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 232 β1  PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE TriggerVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVP LoopEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL (short)SNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLS β2AHCSCDSRDNTLQVDINGFTGTSTYTSGDQKTIKSTRK C4SKHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA LEPLPIVYYVGRKPKVEQLSNMIVRSCKCSproTGF-  SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT 233 β3arm1VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTE C7SSEYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPL TILYYVGRTPKVEQLSNMVVKSCKCS TGF-  LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 234 β1arm3PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE C4SIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKN (LAP)RTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQG GQRKKR TGF-  SLSLSTSTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPT 235 β3arm1VMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTE C7SSEYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTS (LAP)ELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHG MNRPFLLLMATPLERAQHLQSSRHRRTGF-β1   LSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVP 236 Trigger PGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKE LoopVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVP (short) EPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYL β2 SNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLS C4SAHCSCDSRDNTLQVDINGFTGTSTYTSGDQKTIKSTRK (LAP)KHGMNRPFLLLMATPLERAQHLQSSRHRR

In some embodiments, chimeric proteins may comprise one or more proteinmodules from TGF-β2. Although the crystal structure for the TGF-β2growth factor has been elucidated (Daopin, S. et al., Crystal structureof transforming growth factor-β2: an unusual fold for the superfamily.Science. 1992. 257(5068):369-73) activation mechanisms remain to befully understood. Activation may be dependent upon one or moreinteractions between the TGF-β2 trigger loop and α₉β₁ integrin. TheTGF-β2 trigger loop may comprise similar structural and/or functionalfeatures associated with RGD sequences. TGF-β2 trigger loops may bindintegrins, including, but not limited to α₉β₁ integrins.

According to mouse tissue staining, integrin subunit α₉ is widelyexpressed in skeletal and cardiac muscle, visceral smooth muscle,hepatocytes, airway epithelium, squamous epithelium, choroid plexusepithelium and also on neutrophils (Palmer, E. L. et al., Sequence andtissue distribution of the integrin α₉ subunit, a novel partner of β₁that is widely distributed in epithelia and muscle. Journal of CellBiology. 1993. 123(5):1289-97.) Expression of α₉ is not detected earlierthan E12.5, suggesting that it does not play a major role in theearliest tissue morphogenesis (Wang, A. et al., Expression of theintegrin subunit α₉ in the murine embryo. Developmental Dynamics. 1995.204:421-31.) In vivo functions of α₉ are unclear. Phenotypes observed inknockout mice suggest a role in lymphatic valve development (Bazigou, E.et al., Integrin-α₉ is required for fibronectin matrix assembly duringlymphatic valve morphogenesis. Dev Cell. 2009 Aug. 17 (2):175-86.)Reported interaction partners of integrin α₉β₁ include VCAM-1, the thirdFnIII domain on tenascin C, osteopontin, polydom/SVEP1, VEGF-A and NGF(Yokasaki, Y. et al., Identification of the ligand binding site for theintegrin α₉β₁ in the third fibronectin type III repeat of tenascin C.The Journal of Biological Chemistry. 1998. 273(19):11423-8;Marcinkiewicz, C. et al., Inhibitory effects of MLDG-containingheterodimeric disintegrins reveal distinct structural requirements forinteraction of the integrin α₉β₁ with VCAM-1, tenascin-C, andosteopontin. JBC. 2000. 275(41):31930-7; Oommen, S. et al., Vacularendothelial growth factor A (VEGF-A) induces endothelial and cancer cellmigration through direct binding to integrin α₉β₁. JBC. 2011.286(2):1083-92; Sato-Nishiuchi, R. et al., Polydom/SVEP1 is a ligand forintegrin α₉β₁. JBC. 2012. 287(30):25615-30; Staniszewska, I. et al.,Integrin α₉β₁ is a receptor for nerve growth factor and otherneurotrophins. Journal of Cell Science. 2007. 121(Pt 4):504-13;Yokosaki, Y. et al., The integrin α₉β₁ binds to a novel recognitionsequence (SVVYGLR; SEQ ID NO: 238) in the thrombin-cleavedamino-terminal fragment of osteopontin. JBC. 1999. 274(51):36328-34.)

Binding sites on proteins that interact with α₉β₁ have been mapped usinglinear peptides. These sites include binding sites on tenascin C(AEIDGIEL; SEQ ID NO: 237), osteopontin (SVVYGLR; SEQ ID NO: 238),polydom/SVEP1 (EDDMMEVPY; SEQ ID NO: 239) and VEGF-A (EYP). Unlike α₄β₁and α₅β₁, α₉β₁ does not require a canonical RGD sequence motif. Some,but not all reported targets have an acidic residue/hydrophobicresidue/proline motif. Some also comprise a tyrosine residue.

The trigger loop of TGF-β1 and TGF-β3 carries an RGD sequence whereα_(v)β₆ and/or α_(v)β₈ bind to enable growth factor release. The TGF-β2trigger loop region is different from those of TGF-β1 and TGF-β3,comprising the sequence FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP (SEQ ID NO: 65),without an RGD trimer. Of this region, residues AGIDGTST (SEQ ID NO:240) align with the peptide on the third FnIII domain of tenascin-C thathas been mapped as an α₉β₁ binding site. Also, the tyrosine followingthis region may play a role in potential α₉β₁ binding. Therefore, α₉β₁binding to TGF-β2 could be physiologically relevant. In someembodiments, chimeric proteins of the present invention may comprisetrigger loop sequences comprising any of the sequences listed in Table15.

TABLE 15 Trigger loop sequences SEQ Source ID proteinTrigger loop sequence NO TGF-β2 FAGIDGTSTYTSGDQKTIKSTRKKNSGKTP  65TGF-β2 AGIDGTST 240 TGF-β2  GTSTYTSGDQKTIKSTRKK 180 (short) TGF-β1INGFTTGRRGDLATIHGMNRP 241 TGF-β1 SGRRGDLATI 242 TGF-β1 TGRRGDLATI 243TGF-β3 FKGVDNEDDHGRGDLGRLKKQKDHHNP 244 GDF-8 PGEDGLNP 245 GDF-11PGAEGLHP 246 Inhibin A RPEATP 247 BMP-9 SHRKGCDTLDISVPPGSRNLP 248 BMP-2RHVRISRSLHQDEHSWSQIRP 249 BMP-4 QHVRISRSLPQGSGNWAQLRP 250 BMP-7IGRHGPQNKQP 251 BMP-6 VGRDGPYDKQP 252 BMP-8 LGQRAPRSQQP 253 Lefty 1RFASQGAPAGLGEP 254 osteopontin SVVYGLR 238 tenascin C AEIDGIEL 237polydom/SVEP1 EDDMMEVPY 239 VEGF-A EYP —

In some embodiments, chimeric proteins of the present invention maycomprise one or more TGF-β2 trigger loops. Such chimeric proteins mayexhibit activation (e.g. growth factor release) regulated in a mannersimilar to that of TGF-β2. Some chimeric proteins of the presentinvention may comprise TGF-β-related proteins wherein one or moreprotein modules are substituted with one or more protein modulescomprising one or more TGF-β2 trigger loops. Some chimeric proteinscomprise TGF-β-related proteins wherein one or more protein modulescomprising at least one RGD sequence are substituted with one or moreprotein modules comprising one or more TGF-β2 trigger loops. In otherembodiments, chimeric proteins may comprise TGF-β1 and/or TGF-β3proteins wherein one or more protein modules comprising at least one RGDsequence are substituted with one or more protein modules comprising oneor more TGF-β2 trigger loops. Such chimeric proteins may exhibit TGF-β1activity.

In some embodiments, chimeric proteins of the present invention maycomprise one or more protein modules from BMPs. Protein modulescomprising sequences from BMPs may comprise sequences from any of thoseBMP modules disclosed in FIGS. 8A-8G. Chimeric proteins of the presentinvention comprising one or more BMP protein module may be useful forthe development of antibodies and/or assays to study, enhance and/orperturb BMP interactions with other proteins, including, but not limitedto RGM proteins.

Chimeric proteins may comprise detectable labels. Detectable labels maybe used to allow for detection and/or isolation of chimeric proteins.Such detectable labels may comprise biotin labels, polyhistidine tagsand/or flag tags. Tags may be used to identify and/or isolate taggedproteins. Proteins produced may comprise additional amino acids encodingone or more 3C protease cleavage site. Such sites allow for cleavage atthe 3C protease cleavage site upon treatment with 3C protease,including, but not limited to rhinovirus 3C protease. 3C proteasecleavage sites may be introduced to allow for removal of detectablelabels from chimeric proteins.

Protein Expression

In some embodiments, synthesis of recombinant proteins of the presentinvention may be carried out according to any method known in the art.Some protein synthesis may be carried out in vitro. Some proteinsynthesis may be carried out using cells. Such cells may be bacterialand/or eukaryotic. In some embodiments, eukaryotic cells may be used forprotein synthesis. Some such cells may be mammalian. Some mammaliancells used for protein expression may include, but are not limited tomouse cells, rabbit cells, rat cells, monkey cells, hamster cells andhuman cells. Such cells may be derived from a cell line. In otherembodiments, human cells may be used. In further embodiments, cell linesmay include, but are not limited to HEK293 cells, CHO cells, HeLa cells,Sw-480 cells, EL4 T lymphoma cells, TMLC cells, 293T/17 cells, Hs68cells, CCD1112sk cells, HFF-1 cells, Keloid fibroblasts, A204 cells, L17RIB cells and C₂C₁₂ cells.

In some embodiments, 293 cells are used for synthesis of recombinantproteins of the present invention. These cells are human cells thatpost-translationally modify proteins with human-like structures (e.g.glycans). Such cells are easily transfectable and scalable and are ableto grow to high densities in suspension culture. 293 cells may include293E cells. 293E cells are HEK293 cells stably expressing EBNA1(Epstein-Barr virus nuclear antigen-1). In some cases, 293E cells may begrown in serum-free medium to simplify down-stream purification. In somecases, 293-6E cells (NRC Canada, Ottawa, CA) may be used. Such cellsexpress truncated EBNA1 (EBNA1t) and may comprise enhanced production ofrecombinant proteins and may be optimized for growth and/or proteinexpression in serum-free medium to simplify down-stream purification. Insome cases, insect cells may be used to express recombinant proteins ofthe invention. In some cases, insect cell expression may be carried outusing Spodoptera frugiperda cells including, but not limited to Sf9and/or Sf-21 cells. In some cases, insect cell cultures may compriseTrichoplusia ni cells, including, but not limited to Tn-368 and/orHIGH-FIVE™ BTI-TN-5B1-4 cells. A further list of exemplary insect celllines can be found in U.S. Pat. No. 5,024,947, the contents of which areherein incorporated by reference in their entirety.

In some embodiments, recombinant proteins of the invention may comprisean antibody Fc domain to create an Fc fusion protein. The formation ofan Fc fusion protein with any of the recombinant proteins describedherein may be carried out according to any method known in the art,including as described in Czajkowsky, D. M. et al., 2012. EMBO Mol Med.4(10):1015-28 and U.S. Pat. Nos. 5,116,964, 5,541,087 and 8,637,637, thecontents of each of which are herein incorporated by reference in theirentirety. Fc fusion proteins of the invention may be linked to the hingeregion of an IgG Fc via cysteine residues in the Fc hinge region.Resulting Fc fusion proteins may comprise an antibody-like structure,but without C_(H1) domains or light chains. In some cases, Fc fusionproteins may comprise pharmacokinetic profiles comparable to nativeantibodies. In some cases, Fc fusion proteins of the invention maycomprise an extended half-life in circulation and/or altered biologicalactivity. In some cases, Fc fusion proteins of the invention may beprepared using any of the TGF-β family proteins or TGF-β-relatedproteins described herein. In some cases, Fc fusion proteins maycomprise TGF-β, GDF-8 and/or GDF-11.

Sequences encoding recombinant proteins of the present invention may beinserted into any number of DNA vectors known in the art for expression.Such vectors may include plasmids. In some embodiments, sequencesencoding recombinant proteins of the present invention are cloned intopTT5 vectors (NRC Biotechnology Research Institute, Montreal, Québec.)In other embodiments pTT22, pTT28, pYD5, pYD7, pYD11 (NRC BiotechnologyInstitute, Montréal, Québec) and/or pMA vectors (Life Technologies,Carlsbad, Calif.) may be used. Vectors may comprise promoter sequencesto modulate expression of sequences encoding recombinant proteins of thepresent invention. Such promoters may be constitutively active and/ormay be regulated by extrinsic and/or intrinsic factors. Some extrinsicfactors may be used to enhance or suppress expression of sequencesencoding recombinant proteins of the present invention. Some vectors mayencode nuclear localization signals that may be incorporated intorecombinant proteins of the present invention upon translation. Somevectors may produce mRNA transcripts that comprise nuclear exportsignals. RNA transcribed from a modified pTT5 vector (pTT5-WPRE)contains an element that facilitates nuclear export of the transcripts.Some vectors may be modified by insertion of one or moreligation-independent cloning (LIC) cassettes to provide for simplercloning.

Vectors encoding recombinant proteins of the present invention may bedelivered to cells according to any method known in the art, including,but not limited to transfection, electroporation and/or transduction. Insome embodiments, vectors may comprise one or more elements to enhancevector replication in host cells. In some embodiments, vectors maycomprise oriP sites for episomal replication in cells that expressEBNA-1.

In some cases, cells are stably transfected to produce recombinantproteins of the present invention. Stably transfected cells passtransfected genes to daughter cells during cell division, thuseliminating the need for repeated transfection. In some cases, thetransfected genes are stably inserted into the genome of the transfectedcells. Transfected genes may comprise genes for cell selection, such asgenes that confer resistance to one or more toxic or repressivecompounds. Such genes may be used to support the growth of only cellswith stable incorporation of the transfected genes when grown in thepresence of such one or more toxic or repressive compounds (e.g.puromycin, kenomycin, etc.) Cell selection may also comprise selectingcells based on overall recombinant protein expression levels.Determination of such levels may be carried out, for example, by WesternBlot and/or ELISA.

In some embodiments, nucleotide sequences encoding recombinant proteinsof the present invention may comprise one or more woodchuck hepatitisvirus posttranscriptional regulatory element (WPRE). RNA nucleic acidscomprising such elements may comprise the sequenceGCCACGGCGGAACUCAUCGCCGCCUGCCUUGCCCGCUGCUGGACAGGGGCUCGGCUGUUGGGCACUGACAAUUCCGUGGU (SEQ ID NO: 255). RNA comprising WPREs may betranscribed from DNA comprising the sequenceAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO: 256). WPREs may enhance translation ofnucleic acids comprising WPREs. Such enhanced translation may be due toincreased cytoplasmic export of newly transcribed mRNA.

In some embodiments, recombinant proteins may comprise one or moresecretion signal sequences. As used herein, the term “secretion signalsequence” refers to a chain of amino acids (or nucleotides that encodethem at the nucleic acid level) that when part of a protein, modulatesecretion of such proteins from cells. Some secretion signal sequencesmay be located at protein termini. In other embodiments, secretionsignal sequences may be N-terminal amino acid sequences. Othersecretions signal sequences may comprise the secretion signal of the Igkappa chains. Such Ig kappa chains may be human Ig kappa chains. In someembodiments, secretion signal sequences may comprise the amino acidsequence MDMRVPAQLLGLLLLWFSGVLG (SEQ ID NO: 257).

In some embodiments, recombinant proteins of the present invention mayrequire coexpression with one or more other proteins for properexpression, folding, secretion, activity and/or function. Somerecombinant GPCs of the present invention may be coexpressed with LTBPs,fibrillins and/or GARP.

In some embodiments, recombinant proteins of the present invention maybe biotinylated. As used herein, the term “biotinylating” refers to theattaching of one or more biotin labels. Such biotin labels mayfacilitate interactions of biotinylated recombinant proteins with avidinand/or streptavidin coated surfaces and/or proteins. As used herein, a“biotin label” refers to a detectable label comprising one or morebiotin molecules. The term “biotinylated” refers to a molecule orprotein that comprises one or more biotin labels. Biotin molecules bindwith high affinity to avidin and streptavidin molecules. This propertymay be used to capture biotinylated proteins using avidin and/orstretavidin coated materials. Some recombinant GPCs of the presentinvention may be biotinylated near the N-terminus. Such recombinant GPCsmay be introduced to avidin/streptavidin coated cell culture surfaces,allowing biotinylated recombinant GPCs to adhere to the surface in amanner such that the orientation and bonding of such bound GPCs mimicsthe orientation and bonding of GPCs to LTBPs, fibrillins and/or GARPs.

In some embodiments, recombinant proteins produced may be analyzed forquality control purposes to assess both biophysical properties as wellas bioactive properties. Biophysical characterization may includeassessing protein migration patterns after reducing and/or non-reducingSDS PAGE. Biophysical characterization may also comprise gel filtration,mass spectrometric analysis and/or analysis of association/dissociationbetween LAPs or LAP-like domains and growth factor domains. Bioactiveproperties may be analyzed by assessing reactivity with antibodiesand/or signaling activity of dissociated growth factors and/or latentGPCs.

Some proteins produced may comprise additional amino acids encoding oneor more detectable labels for purification [e.g. polyhistidine tag, flagtag, etc.] In some embodiments, proteins are N-terminally labeled. Insome embodiments, proteins are C-terminally labeled. In someembodiments, proteins are biotinylated. In some embodiments, recombinantproteins of the present invention are N-terminally biotinylated.

Proteins produced may comprise additional amino acids encoding one ormore 3C protease cleavage site. Such sites allow for cleavage betweenresidues Q and G of the 3C protease cleavage site upon treatment with 3Cprotease, including, but not limited to rhinovirus 3C protease. In someembodiments, such cleavage sites are introduced to allow for removal ofdetectable labels from recombinant proteins.

In some embodiments, modification of expressed growth factor proproteinsmay be carried out by enzymatic cleavage. In some cases, proproteinconvertases may be used. Such proprotein convertases may include, butare not limited to furin/PACE3, PC1/3, PC2, PC4, PC5/6, PACE4 and PC7.Proprotein convertase cleavage may be caried out in solution or intissue culture. In some cases, proprotein convertases are expressed incells expressing proproteins to be cleaved. In some cases, proproteinconvertases are added to tissue cultures of cells expressing proproteinsto be cleaved.

Antibodies

In some embodiments, compounds and/or compositions of the presentinvention may comprise antibodies or fragments thereof. As used herein,the term “antibody” is referred to in the broadest sense andspecifically covers various embodiments including, but not limited tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g. bispecific antibodies formed from at least two intact antibodies),and antibody fragments such as diabodies so long as they exhibit adesired biological activity. Antibodies are primarily amino-acid basedmolecules but may also comprise one or more modifications (including,but not limited to the addition of sugar moieties, fluorescent moieties,chemical tags, etc.)

Recombinant and Chimeric Protein Use in Antibody Generation

In some embodiments, recombinant and/or chimeric proteins describedherein may be used as antigens (referred to herein as antigenicproteins) to generate antibodies. Such antigenic proteins may compriseepitopes that may be less accessible for antibody generation in similarwild type proteins. Some antibodies directed to antigenic proteins ofthe present invention may modulate the release of one or more growthfactors from one or more GPCs.) Some such antibodies may be stabilizing[reducing or preventing dissociation between two agents, (e.g.growth-factor release from GPCs, GPC release from one or more proteininteractions)] and/or releasing [enhancing the dissociation between twoagents (e.g. growth-factor release from GPCs, GPC release from one ormore protein interactions)] antibodies. Antigenic proteins of thepresent invention may comprise TGF-β-related proteins as well ascomponents and/or protein modules thereof. In some cases, antigenicproteins of the present invention may comprise prodomains withoutassociated growth factors, furin cleavage-deficient mutants, mutantsdeficient in extracellular protein associations and/or combinationsthereof.

In some embodiments, antigenic proteins may comprise TGF-β-relatedproteins and/or modules thereof. Such antigenic proteins may compriseepitopes from regions where growth factors associate with or comprisestereological proximity with prodomain regions. Antibodies of thepresent invention directed to such epitopes may bind overlapping regionsbetween growth factors and prodomains. Such antibodies maystereologically inhibit the dissociation of growth factors from GPCs.

In some embodiments, antigenic proteins comprise only the prodomain oronly the growth factor from a particular GPC. Epitopes present on suchantigenic proteins may be shielded or unexposed in intact GPCs. Someantibodies of the present invention may be directed to such epitopes.Such antibodies may be releasing antibodies, promoting growth factordissociation from GPCs. Further antibodies may compete with free growthfactor for prodomain binding, thereby promoting growth factordissociation from GPCs.

In some embodiments, antigenic proteins may comprise proproteinconvertase (e.g. furin) cleavage site mutations. Such mutations mayprevent enzymatic cleavage of growth factors from their prodomains. Someantibodies of the present invention may be directed to epitopes presenton such mutant proteins. Such antibodies may stabilize the associationbetween prodomains and growth factors. In some embodiments, furincleavage site mutants comprise D2G mutants as described herein.

In some embodiments, antigenic proteins comprising prodomains maycomprise N-terminal mutations that lead to decreased prodomainassociation with LTBPs and/or GARP and therefore may present epitopes inthe N-terminal region that may otherwise be shielded by thoseassociations. Some antibodies of the present invention may be directedto such epitopes. Some antigenic proteins comprising TGF-β1 prodomainsmay comprise C4S mutations. Such mutations may prevent association ofantigenic proteins with LTBPs and/or GARP, making these proteins usefulfor presenting N-terminal epitopes. Antibodies directed to C4S mutantsmay prevent GPC association with LTBPs and/or GARP. Some antibodiesdirected to C4S mutants may reduce growth factor signaling in aparticular niche. Some such antibodies may reduce or prevent the releaseof growth factor by blocking the ability of the GPCs to associatesecurely with the extracellular matrix.

In some embodiments, antigenic proteins may comprise one or morerecombinant LTBP. Such recombinant LTBPs may comprise LTBP1, LTBP2,LTBP3, LTBP4, alternatively spliced variants and/or fragments thereof.Recombinant LTBPs may also be modified to comprise one or moredetectable labels. Such detectable labels may include, but are notlimited to biotin labels, polyhistidine tags, myc tags, HA tags and/orfluorescent tags.

In some embodiments, antigenic proteins may comprise one or morerecombinant protein and/or chimeric protein complexed with one or morerecombinant LTBP. Some antigenic proteins may comprise proproteinconvertase cleavage site mutants (e.g. D2G mutants, AXXA mutants)complexed with one or more recombinant LTBP. Some such recombinant LTBPsmay comprise LTBP1S. Some recombinant LTBPs may comprise one or moredetectable labels, including, but not limited to biotin labels,polyhistidine tags and/or flag tags.

In some embodiments, antigenic proteins may comprise GARP (or homologuesthereof, including, but not limited to LRRC33). Such GARP may berecombinant, referred to herein as recombinant GARP. Some recombinantGARPs may comprise one or more modifications, truncations and/ormutations as compared to wild type GARP. Recombinant GARPs may bemodified to be soluble. In other embodiments, recombinant GARPs aremodified to comprise one or more detectable labels. In furtherembodiments, such detectable labels may include, but are not limited tobiotin labels, polyhistidine tags, flag tags, myc tags, HA tags and/orfluorescent tags. In some embodiments, antigenic proteins may compriseone or more recombinant protein and/or chimeric protein complexed withone or more recombinant GARP. In some embodiments, antigenic proteinscomprise LAPs (e.g. TGF-β LAPs) and/or LAP-like domains complexed withrecombinant GARP. In some embodiments, antigenic proteins comprise D2Gmutants (e.g. TGF-β D2G mutants) complexed with recombinant GARP. Insome embodiments, complexed recombinant GARPs may be soluble forms ofGARP (sGARP). In some embodiments, sGARPs comprises one or more biotinlabels, polyhistidine tags and/or flag tags.

In some embodiments, GARPs complexed with LAP and/or LAP-like domainsare desired as antigens, in assays and/or for antibody development. Insuch embodiments, LAPs and/or LAP-like domains may comprise CEDmutations. Such LAPs and/or LAP-like domains may be expressed as GPCs tofacilitate proper protein folding, conformation and/or expression, butthe CED mutations present may enhance growth factor release, leaving thedesired GARP-LAP (or LAP-like domain) complex behind. GARP-LAP (orLAP-like domain) complexes may be useful as antigens in the productionof releasing antibodies that specifically target GARP-associated GPCs.

In some embodiments, GPCs comprising CED mutations may act to stabilizea natively populated conformation of LAP (or LAP-like domain)characterized by reduced growth factor association (both as a free LAPor LAP-like domains and/or as a GARP and/or LTBP/LAP complex), therebyexposing epitopes that may be less exposed in wild-type proteins. Suchmutations may shift the conformational equilibrium of LAP or LAP-likedomains to facilitate the production of activating antibodies.

In some embodiments, antigenic proteins of the present invention maycomprise one or more protein modules from GDFs (e.g. GDF-11 and/orGDF-8). In some embodiments, antibodies of the present invention may bedirected toward antigenic proteins comprising GDF-8 protein modules. Insome embodiments, such antibodies may modulate GDF-8 levels and/oractivity in one or more niches. In some embodiments, antibodies of thepresent invention may prevent the release of GDF-8 growth factors fromGPCs. In some embodiments, antibodies of the present invention may beused to repair and/or enhance muscle tissues.

In some embodiments, recombinant proteins (including, but not limited tochimeric proteins) described herein may be used in studies to identifyand map epitopes that may be important targets for antibody development.Such studies may be used to identify epitopes that may promote growthfactor release or stabilization of GPCs upon antibody binding.

Releasing Antibodies

As used herein, the term “releasing antibody” refers to an antibody thatincreases the ratio of active and/or free growth factor relative toinactive and/or prodomain-associated growth factor upon the introductionof the antibody to a GPC, cell, niche, natural depot or any other siteof growth factor sequestration. In this context, releasing antibodiesmay be characterized as agonists. As used herein, the term “naturaldepot” refers to a location within a cell, tissue or organ whereincreased levels of a biomolecule or ion are stored. For example, theextracellular matrix may act as a natural depot for one or more growthfactors.

The contact necessary for growth-factor release may be defined as director indirect contact of antibody with a GPC or a component thereof orwith a cellular structure such as an extracellular and/or cellularmatrix protein and/or protein associated with the extracellular and/orcellular matrix [e.g. LTBPs (e.g. LTBP1, LTBP2, LTBP3 and/or LTBP4),fibrillins (e.g. fibrillin-1, fibrillin-2, fibrillin-3 and/orfibrillin-4) perlecan, decorin, elastin, collagen and/or GARPs (e.g.GARP and/or LRRC33)] for release of growth factor. Release of at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of growth factoris sufficient to characterize antibodies of the present invention asreleasing antibodies. It is understood that growth factor release afterantibody administration may be local and may occur over a sustainedperiod of time and may include peaks or spikes of release. Antibodies ofthe present invention may act to release one or more growth factor overminutes, hours, days or longer.

Release profiles may have an initial peak or burst within from about 4hours to about 7 days of contacting in vivo or shorter periods in vitro.For example, initial peak or burst may occur from about 4 hours to about5 hours, or from about 4 hours to about 6 hours, or from about 4 hoursto about 7 hours, or from about 4 hours to about 8 hours, or from about4 hours to about 9 hours, or from about 4 hours to about 10 hours, orfrom about 4 hours to about 11 hours, or from about 4 hours to about 12hours, or from about 4 hours to about 24 hours, or from about 4 hours toabout 36 hours, or from about 4 hours to about 48 hours, or from about 1day to about 7 days, or from about 1 day to about 2 days, or from about1 day to about 3 days, or from about 1 day to about 4 days, or fromabout 4 days to about 5 days, or from about 4 days to about 6 days, orfrom about 4 days to about 7 days. Compounds and/or compositions of thepresent invention may stimulate the release of 5 to 100% of the growthfactor present. For example, the percent of growth factor release may befrom about 5% to about 10%, or from about 5% to about 15%, or from about5% to about 20%, or from about 5% to about 25%, or from about 10% toabout 30%, or from about 10% to about 40%, or from about 10% to about50%, or from about 10% to about 60%, or from about 20% to about 70%, orfrom about 20% to about 80%, or from about 40% to about 90%, or fromabout 40% to about 100%.

Releasing antibodies generated according to methods described herein maybe generated to release growth factors from GPCs comprising any of thepro-proteins listed in Table 1. In some cases, releasing antibodies aredirected to GPCs comprising TGF-β isoforms and/or one or more modules ofsuch isoforms. In some cases, releasing antibodies are directed to GPCscomprising GDFs and/or one or more modules from GDFs.

Stabilizing Antibodies

As used herein, the term “stabilizing antibody” refers to an antibodythat decreases the ratio of active and/or free growth factor relative toinactive and/or prodomain-associated growth factor upon the introductionof the antibody to one or more GPC, cell, niche, natural depot and/orany other site of growth factor sequestration. In this context,antibodies may be characterized as antagonists. As used herein, an“antagonist” is one which interferes with or inhibits the physiologicalaction of another. Antagonist action may even result in stimulation oractivation of signaling downstream and hence may act agonisticallyrelative to another pathway, separate from the one being antagonized.Pathways are interrelated, so, in one nonlimiting example, a TGF-βantagonist could act as a BMP agonist and vice versa. In the context ofcellular events, as used herein, the term “downstream” refers to anysignaling or cellular event that happens after the action, binding ortargeting by compounds and/or compositions of the present invention.

Contact necessary for inhibition or stabilization may be direct orindirect contact between antibody and GPC or components thereof or withcellular structures such as an extracellular and/or cellular matrixprotein and/or protein associated with the extracellular and/or cellularmatrix [e.g. LTBPs (e.g. LTBP1, LTBP2, LTBP3 and/or LTBP4), fibrillins(e.g. fibrillin-1, fibrillin-2, fibrillin-3 and/or fibrillin-4)perlecan, decorin, elastin, collagen and/or GARPs (e.g. GARP and/orLRRC33)] whereby release of growth factor is inhibited. Inhibition ofrelease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore of growth factors may be sufficient, in some cases, to characterizeantibodies of the present invention as inhibitory or stabilizing.Inhibitory antibodies may stabilize GPCs and trap them as heterodimers.

It is understood that inhibition of growth factor release after contactwith one or more antibodies of the present invention may be local andmay occur over a sustained period of time and may include peaks, troughsor spikes. Inhibitory antibodies which may also function to stabilizeGPCs may be defined by their release kinetics. Release of growth factorand corresponding release kinetics, even locally, may be directlymeasured or inferred by downstream signaling events. In someembodiments, changes in protein or nucleic acid concentrations orphenotypic responses may be indicative of the effects of compoundsand/or compositions of the present invention.

Antibodies of the present invention may act to inhibit release of agrowth factor over minutes, hours or days. Inhibition and/orstabilization profiles may have an initial trough within from about 4hours to about 7 days of introduction in vivo or shorter periods invitro. For example, initial trough of inhibition or stabilization mayoccur from about 4 hours to about 5 hours, or from about 4 hours toabout 6 hours, or from about 4 hours to about 7 hours, or from about 4hours to about 8 hours, or from about 4 hours to about 9 hours, or fromabout 4 hours to about 10 hours, or from about 4 hours to about 11hours, or from about 4 hours to about 12 hours, or from about 4 hours toabout 24 hours, or from about 4 hours to about 36 hours, or from about 4hours to about 48 hours, or from about 1 day to about 7 days, or fromabout 1 day to about 2 days, or from about 1 day to about 3 days, orfrom about 1 day to about 4 days, or from about 4 days to about 5 days,or from about 4 days to about 6 days, or from about 4 days to about 7days. Introduction of compounds and/or compositions of the presentinvention may lead to inhibition and/or stabilization of 5% to 100% ofgrowth factor present. For example, the percent of growth factorinhibition or stabilization may be from about 5% to about 10%, fromabout 5% to about 15%, from about 5% to about 20%, from about 5% toabout 25%, from about 10% to about 30%, from about 10% to about 40%,from about 10% to about 50%, from about 10% to about 60%, from about 20%to about 70%, from about 20% to about 80%, from about 40% to about 90%or from about 40% to about 100%.

Stabilizing antibodies generated according to methods described hereinmay be generated to block the release of growth factors from GPCscomprising any of the pro-proteins listed in Table 1. Such antibodiesmay physically interact with GPC protease cleavage sites and/or blockthe interaction of proteolytic enzymes that may target such cleavagesites. In some cases, stabilizing antibodies are directed to GPCscomprising TGF-β isoforms and/or one or more modules of such isoforms.In some cases, stabilizing antibodies are directed to GPCs comprisingGDFs and/or one or more modules from GDFs.

Stabilizing antibodies directed to GPCs comprising GDF-8 may blockmetalloproteinase cleavage of such complexes. Such agents may bind toGPCs comprising GDF-8 in such a way as to physically preventinteractions between such GPCs and metalloproteinases targeting suchGPCs. Agents that actually target metalloproteinases themselves havebeen described previously (see U.S. Pat. No. 7,572,599, the contents ofwhich are herein incorporated by reference in their entirety.)

Antibody Selection

A desired antibody may be selected from a larger pool of two or morecandidate antibodies based on the desired antibody's ability toassociate with desired antigens and/or epitopes. Such antigens and/orepitopes may include, but are not limited to any of those describedherein, including, but not limited to recombinant proteins, chimericproteins, GPCs, prodomains (e.g. LAPs or LAP-like domains), growthfactors, protein modules, LTBPs, fibrillins, GARP, TGF-β-relatedproteins and/or mutants and/or variants and/or complexes and/orcombinations thereof. Selection of desired antibodies may be carried outusing an antibody binding assay, such as a surface Plasmonresonance-based assay, an enzyme-linked immunosorbent assay (ELISA) orfluorescence-associated cell sorting (FACS)-based assay. Such assays mayutilize a desired antigen to bind a desired antibody and then use one ormore detection methods to detect binding.

In some embodiments, antibodies of the present invention may be selectedfrom a larger pool of two or more candidate antibodies based on theirability to associate with desired antigens and/or epitopes from multiplespecies (referred to herein as “positive selection.”)

In some embodiments, such species may comprise vertebrate species. Insome embodiments, such species may comprise mammalian species. In someembodiments, such species may include, but are not limited to mice,rats, rabbits, goats, sheep, pigs, horses, cows and/or humans.

In some embodiments, negative selection is used to remove antibodiesfrom a larger pool of two or more candidate antibodies. As used hereinthe term “negative selection” refers to the elimination of one or morefactors from a group based on their ability to bind to one or moreundesired antigens and/or epitopes. In some embodiments, undesiredantigens and/or epitopes may include, but are not limited to any ofthose described herein, including, but not limited to recombinantproteins, chimeric proteins, GPCs, prodomains (e.g. LAPs or LAP-likedomains), growth factors, protein modules, LTBPs, fibrillins, GARPs,TGF-β-related proteins and/or mutants and/or variants and/orcombinations and/or complexes thereof.

In some embodiments, antibodies of the present invention may be directedto prodomains (e.g. the prodomain portion of a GPC and/or free LAP orLAP-like domains) that decrease growth factor signaling and/or levels(e.g. TGF-β growth factor signaling and/or levels) in a given niche. Insome embodiments, antibodies of the present invention may directed toLAPs or LAP-like domains that increase growth factor signaling and/orlevels in a given niche. In some embodiments, antibodies of the presentinvention may be directed to prodomains (e.g. LAPs or LAP-like domains)and/or GPCs only when complexed with LTBPs, fibrillins and/or GARP.

In some embodiments, antibodies of the present invention may be selectedfrom a larger pool of two or more candidate antibodies based on theirability to modulate growth factor levels and/or activity. In some cases,growth factor activity assays may be used to test the ability ofcandidate antibodies to modulate growth factor activity. Growth factoractivity assays may include, cell-based assays as described hereinbelow.Additional assays that may be used to determine the effect of candidateantibodies on growth factor activity may include, but are not limited toenzyme-linked immunosorbent assay (ELISA), Western blotting, reporterassays (e.g. luciferase-based reporter assays or other enzyme-basedreporter assays), PCR analysis, RT-PCR analysis and/or other methodsknown in the art including any of the methods described in U.S.Provisional Patent Applications 61/722,919, filed Nov. 6, 2012 and61/722,969, filed Nov. 6, 2012, the contents of each of which are hereinincorporated by reference in their entireties.

In some embodiments, one or more recombinant proteins or antibodiesdisclosed herein may be used in assays to test, develop and/or selectantibodies. Recombinant GPCs may be expressed to test releasing and/orstabilizing abilities of one or more antibodies being assayed. In someembodiments, recombinant proteins may be expressed as positive ornegative control components of assays. In some embodiments, multiplerecombinant proteins may be expressed at once to modulate growth factorrelease and/or activity, wherein such recombinant proteins may actsynergistically or antagonistically in such modulation.

In some embodiments GPCs comprising CED mutations may provide a baselinelevel of growth factor activity in assays designed to test releasingantibodies, as these mutant proteins are sufficient for producing abiological effect in humans. In some embodiments, GPCs comprising CEDmutations may be used as positive controls in activity assays gearedtoward screening for releasing antibodies. In some embodiments, GPCscomprising CED mutations may be used for screening for stabilizingantibody activity, as they can be presumably activated in the absence ofintegrins. In such assays, GPCs comprising CED mutations may beexpressed in cell lines (e.g. 293 cells or others) and growth factoractivity and/or release may be assessed in the presence or absence ofantibodies being tested. In some embodiments, co-expression of GPCscomprising CED mutation with wild type GPCs (including, but not limitedto TGF-β1, TGF-β2, or TGF-β3) could also be used to regulate free growthfactor levels. In such embodiments, modulation of free growth factorlevels may accomplished by co-transfection of different ratios of wildtype and mutant GPCs (e.g. 1:1, 1:2, 1:3, 1:4, 1:5, 1:10). In someembodiments, further co-expression of LTBPs, fibrillins or GARPs may becarried out to add one or more additional levels of free growth factormodulation.

Antibody Development

In some embodiments, compounds and/or compositions of the presentinvention comprising antibodies, antibody fragments, their variants orderivatives as described above are specifically immunoreactive withantigenic proteins as described herein.

Antibodies of the present invention may be characterized by their targetmolecule(s), by the antigens used to generate them, by their function(whether as agonists, antagonists, growth-factor releasing, GPCstabilizing, activating and/or inhibitory) and/or by the cell niche inwhich they function.

As used herein the term, “antibody fragment” refers to any portion of anintact antibody. In some embodiments, antibody fragments compriseantigen binding regions from intact antibodies. Examples of antibodyfragments may include, but are not limited to Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite. Also produced is a residual “Fc” fragment, whose name reflects itsability to crystallize readily. Pepsin treatment yields an F(ab′)2fragment that has two antigen-binding sites and is still capable ofcross-linking antigen. Compounds and/or compositions of the presentinvention may comprise one or more of these fragments. For the purposesherein, an “antibody” may comprise a heavy and light variable domain aswell as an Fc region.

As used herein, the term “native antibody” refers to a usuallyheterotetrameric glycoprotein of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

As used herein, the term “variable domain” refers to specific antibodydomains that differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen.

As used herein, the term “Fv” refers to antibody fragments comprisingcomplete antigen-recognition and antigen-binding sites. These regionsconsist of a dimer of one heavy chain and one light chain variabledomain in tight, non-covalent association.

As used herein, the term “light chain” refers to a component of anantibody from any vertebrate species assigned to one of two clearlydistinct types, called kappa and lambda based on amino acid sequences ofconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains, antibodies can be assigned to differentclasses. There are five major classes of intact antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Asused herein, the term “Single-chain Fv” or “scFv” refers to a fusionprotein of V_(H) and V_(L) antibody domains, wherein these domains arelinked together into a single polypeptide chain. In some embodiments,the Fv polypeptide linker enables the scFv to form the desired structurefor antigen binding.

As used herein, the term “bispecific antibody” refers to an antibodycapable of binding two different antigens. Such antibodies typicallycomprise regions from at least two different antibodies. Bispecificantibodies may include any of those described in Riethmuller, G. 2012.Cancer Immunity. 12:12-18, Marvin, J. S. et al., 2005. ActaPharmacologica Sinica. 26(6):649-58 and Schaefer, W. et al., 2011. PNAS.108(27):11187-92, the contents of each of which are herein incorporatedby reference in their entirety.

As used herein, the term “diabody” refers to a small antibody fragmentwith two antigen-binding sites. Diabodies comprise a heavy chainvariable domain V_(H) connected to a light chain variable domain V_(L)in the same polypeptide chain. By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al. (Hollinger,P. et al., “Diabodies”:Small bivalent and bispecific antibody fragments.PNAS. 1993. 90:6444-8) the contents of each of which are incorporatedherein by reference in their entirety.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous cells (orclones), i.e., the individual antibodies comprising the population areidentical and/or bind the same epitope, except for possible variantsthat may arise during production of the monoclonal antibodies, suchvariants generally being present in minor amounts. In contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. The monoclonal antibodies hereininclude “chimeric” antibodies (immunoglobulins) in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies.

As used herein, the term “humanized antibody” refers to a chimericantibody comprising a minimal portion from one or more non-human (e.g.,murine) antibody source with the remainder derived from one or morehuman immunoglobulin sources. For the most part, humanized antibodiesare human immunoglobulins (recipient antibody) in which residues fromthe hypervariable region from an antibody of the recipient are replacedby residues from the hypervariable region from an antibody of anon-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and/orcapacity.

As used herein, the term “hypervariable region” refers to regions withinthe antigen binding domain of an antibody comprising amino acid residuesresponsible for antigen binding. The amino acids present within thehypervariable regions determine the structure of the complementaritydetermining region (CDR). As used herein, the term “CDR” refers toregions of antibodies comprising a structure that is complimentary toits target antigen or epitope.

In some embodiments, compounds and/or compositions of the presentinvention may be antibody mimetics. As used herein, the term “antibodymimetic” refers to any molecule which mimics the function or effect ofan antibody and which binds specifically and with high affinity to theirmolecular targets. In some embodiments, antibody mimetics may bemonobodies, designed to incorporate the fibronectin type III domain(Fn3) as a protein scaffold (U.S. Pat. No. 6,673,901; U.S. Pat. No.6,348,584). In some embodiments, antibody mimetics may be those known inthe art including, but are not limited to affibody molecules, affilins,affitins, anticalins, avimers, Centyrins, DARPINS™, Fynomers and Kunitzand domain peptides. In other embodiments, antibody mimetics may includeone or more non-peptide region.

As used herein, the term “antibody variant” refers to a biomoleculeresembling an antibody in structure and/or function comprising somedifferences in their amino acid sequence, composition or structure ascompared to a native antibody.

The preparation of antibodies, whether monoclonal or polyclonal, isknown in the art. Techniques for the production of antibodies are wellknown in the art and described, e.g. in Harlow and Lane “Antibodies, ALaboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlowand Lane “Using Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory Press, 1999 and “Therapeutic Antibody Engineering: Currentand Future Advances Driving the Strongest Growth Area in thePharmaceutical Industry” Woodhead Publishing, 2012.

Standard Monoclonal Antibody Generation

In some embodiments, antibodies are generated in knockout mice, lackingthe gene that encodes for desired target antigens. Such mice may not betolerized to target antigens and therefore may be better suited forgenerating antibodies against such antigens that may cross react withhuman and mouse forms of the antigen. For the production of monoclonalantibodies, host mice may be immunized with recombinant proteins toelicit lymphocytes that specifically bind such proteins. Resultinglymphocytes may be collected and fused with immortalized cell lines.Resulting hybridoma cells may be cultured in suitable culture mediumwith selection agents to support the growth of only fused cells.

Desired hybridoma cell lines may be identified through bindingspecificity analysis of secreted antibodies for target peptides andclones of such cells may be subcloned through limiting dilutionprocedures and grown by standard methods. Antibodies produced bysubcloned hybridoma cells may be isolated and purified from culturemedium by standard immunoglobulin purification procedures

Recombinant Antibodies

Recombinant antibodies of the present invention may be generatedaccording to any of the methods disclosed in U.S. Provisional PatentApplications 61/722,919, filed Nov. 6, 2012 and 61/722,969, filed Nov.6, 2012, the contents of each of which are herein incorporated byreference in their entireties. In some embodiments, recombinantantibodies may be produced using hybridoma cells produced according tomethods described herein. Heavy and light chain variable region cDNAsequences of antibodies may be determined using standard biochemicaltechniques. Total RNA may be extracted from antibody-producing hybridomacells and converted to cDNA by reverse transcriptase (RT) polymerasechain reaction (PCR). PCR amplification may be carried out on resultingcDNA to amplify variable region genes. Such amplification may comprisethe use of primers specific for amplification of heavy and light chainsequences. Resulting PCR products may then be subcloned into plasmidsfor sequence analysis. Once sequenced, antibody coding sequences may beplaced into expression vectors. For humanization, coding sequences forhuman heavy and light chain constant domains may be used to substitutefor homologous murine sequences. The resulting constructs may then betransfected into mammalian cells for large scale translation.

Development of Cytotoxic Antibodies

In some embodiments, antibodies of the present invention may be capableof inducing antibody-dependent cell-mediated cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC) and/or antibody-dependent cellphagocytosis (ADCP.) ADCC is an immune mechanism whereby cells are lysedas a result of immune cell attack. Such immune cells may include CD56+cells, CD3-natural killer (NK) cells, monocytes and neutrophils (Strohl,W. R. Therapeutic Antibody Engineering. Woodhead Publishing,Philadelphia Pa. 2012. Ch. 8, p 186, the contents of which are hereinincorporated by reference in their entirety.)

In some cases, antibodies of the present invention may be engineered tocomprise a given isotype depending on whether or not ADCC or ADCP isdesired upon antibody binding. Such antibodies, for example, may beengineered according to any of the methods disclosed by Alderson, K. L.et al., J Biomed Biotechnol. 2011. 2011:379123.) In the case of mouseantibodies, different isotypes of antibodies are more effective atpromoting ADCC. IgG2a, for example, is more effective at inducing ADCCthan is IgG2b. Some antibodies of the present invention, comprisingmouse IgG2b antibodies may be reengineered to comprise IgG2a antibodies.Such reengineered antibodies may be more effective at inducing ADCC uponbinding cell-associated antigens.

In some embodiments, genes encoding variable regions of antibodiesdeveloped according to methods of the present invention may be clonedinto mammalian expression vectors encoding human Fc regions. Such Fcregions may comprise Fc regions from human IgG1κ. IgG1κ Fc regions maycomprise amino acid mutations known to enhance Fc-receptor binding andantibody-dependent cell-mediated cytotoxicity ADCC.

In some cases, antibodies may be engineered to reduce ADCC. Antibodiesthat do not activate ADCC or that are associated with reduced levels ofADCC may be desireable for antibody embodiments of the presentinvention, in some cases due to no or limited immune-mediated clearance,allowing longer half-lives in circulation.

Antibody Fragment Display Library Screening Techniques

In some embodiments, antibodies of the present invention may be producedand/or optimized using high throughput methods of discovery. Suchmethods may include any of the display techniques (e.g. display libraryscreening techniques) disclosed in U.S. Provisional Patent Applications61/722,919, filed Nov. 6, 2012 and 61/722,969, filed Nov. 6, 2012, thecontents of each of which are herein incorporated by reference in theirentireties. In some embodiments, synthetic antibodies may be designed,selected or optimized by screening target antigens using displaytechnologies (e.g. phage display technologies.) Phage display librariesmay comprise millions to billions of phage particles, each expressingunique antibody fragments on their viral coats. In some cases, cDNAencoding each fragment may contain the same sequence with the exceptionof unique sequences encoding variable loops of the complementaritydetermining regions (CDRs). V_(H) chains of CDRs may be expressed as afusion protein, linked to viral coat proteins (e.g. the N-terminus ofthe viral pIII coat protein.) V_(L) chains may be expressed separatelyfor assembly with V_(H) chains in the periplasm prior to complexincorporation into viral coats.

For selection, target antigens may be incubated, in vitro, with phagedisplay library particles for precipitation of positive bindingpartners. This process is referred to herein as “phage enrichment.” Insome cases, phage enrichment comprises solid-phase phage enrichment.According to such enrichment, target antigens are bound to a substrate(e.g. by passive adsorption) and contacted with one or more solutionscomprising phage particles. Phage particles with affinity for suchtarget antigens are precipitated out of solution. In some cases, phageenrichment comprises solution-phase phage enrichment where targetantigens are present in a solution that is combined with phagesolutions. According to such methods, target antigens may comprisedetectable labels (e.g. biotin labels) to facilitate retrieval fromsolution and recovery of bound phage.

After selection, cDNA encoding CDRs of precipitated library members maybe sequenced from the bound phage. Such sequences may be directlyincorporated into antibody sequences for recombinant antibodyproduction, or mutated and utilized for further optimization through invitro affinity maturation.

In some cases phage display screening may be used to generate broadlydiverse panels of antibodies. Such diversity may be measured bydiversity of antibody sequences and/or diversity of epitopes targeted.

Affinity Maturation Techniques

Affinity maturation techniques of the present invention may comprise anyof those disclosed in U.S. Provisional Patent Applications 61/722,919,filed Nov. 6, 2012 and 61/722,969, filed Nov. 6, 2012, the contents ofeach of which are herein incorporated by reference in their entireties.After antibody fragments capable of binding target antigens areidentified (e.g. through the use of phage display libraries as describedabove) high affinity mutants may be derived from these through theprocess of affinity maturation. Affinity maturation technology is usedto identify sequences encoding CDRs that have the highest affinity fortarget antigens. Using such technologies, select CDR sequences (e.g.ones that have been isolated or produced according to processesdescribed herein) may be mutated randomly as a whole or at specificresidues to create millions to billions of variants. Such variants maybe subjected to repeated rounds of affinity screening (e.g. displaylibrary screening) for their ability to bind target antigens. Suchrepeated rounds of selection, mutation and expression may be carried outto identify antibody fragment sequences with the highest affinity fortarget antigens. Such sequences may be directly incorporated intoantibody sequences for recombinant antibody production.

Antibody Characterization

Compounds and/or compositions of the present invention comprisingantibodies may act to decrease local concentration of one or more GPCthrough removal by phagocytosis, pinocytosis, or inhibiting assembly inthe extracellular matrix and/or cellular matrix. Introduction ofcompounds and/or compositions of the present invention may lead to theremoval of 5% to 100% of the growth factor present in a given area. Forexample, the percent of growth factor removal may be from about 5% toabout 10%, from about 5% to about 15%, from about 5% to about 20%, fromabout 5% to about 25%, from about 10% to about 30%, from about 10% toabout 40%, from about 10% to about 50%, from about 10% to about 60%,from about 20% to about 70%, from about 20% to about 80%, from about 40%to about 90% or from about 40% to about 100%.

Measures of release, inhibition or removal of one or more growth factorsmay be made relative to a standard or to the natural release or activityof growth factor under normal physiologic conditions, in vitro or invivo. Measurements may also be made relative to the presence or absenceof antibodies. Such methods of measuring growth factor levels, release,inhibition or removal include standard measurement in tissue and/orfluids (e.g. serum or blood) such as Western blot, enzyme-linkedimmunosorbent assay (ELISA), activity assays, reporter assays,luciferase assays, polymerase chain reaction (PCR) arrays, gene arrays,Real Time reverse transcriptase (RT) PCR and the like.

Antibodies of the present invention may bind or interact with any numberof epitopes on or along GPCs or their associated structures to eitherenhance or inhibit growth factor signaling. Such epitopes may includeany and all possible sites for altering, enhancing or inhibiting GPCfunction. In some embodiments, such epitopes include, but are notlimited to epitopes on or within growth factors, regulatory elements,GPCs, GPC modulatory factors, growth factor receiving cells orreceptors, LAPs or LAP-like domains, fastener regions, furin cleavagesites, arm regions, fingers regions, LTBP binding domains, fibrillinbinding domains, glycoprotein A repetitions predominant (GARP) bindingdomains, latency lassos, alpha 1 regions, RGD sequences, bowtie regions,extracellular matrix and/or cellular matrix components and/or epitopesformed by combining regions or portions of any of the foregoing.

Compounds and/or compositions of the present invention exert theireffects via binding (reversibly or irreversibly) to one or more epitopesand/or regions of antibody recognition. While not wishing to be bound bytheory, such binding sites for antibodies, are most often formed byproteins, protein domains or regions. Binding sites may; however,include biomolecules such as sugars, lipids, nucleic acid molecules orany other form of binding epitope.

In some embodiments, antagonist antibodies of the present invention maybind to TGF-β prodomains, stabilizing and preventing integrin-mediatedrelease, for example, by blocking the RGD site or by stabilizing thestructure. Such antibodies would be useful in the treatment ofCamurati-Engelmann disease, in which mutations in the prodomain causeexcessive TGF-β activation. Such antibodies would also be useful inMarfan's syndrome, in which mutations in fibrillins or LTBPs alter TGF-βand BMP activation.

In some embodiments, antibodies of the present invention selectivelyinhibit the release of TGF-β from GPCs associated with LTBPs but notthose associated with GARP. Such antibodies function as anti-fibrotictherapeutics but exhibit minimal inflammatory effects. In someembodiments, GPC-LTBP complex-binding antibodies do not bind GPC-GARPcomplexes. In some embodiments, such antibodies, may not be specific toa particular LTBP or GPC, but may bind to GPCs close to or overlappingwith GARP binding sites, such that binding is impeded by GARP, but notby LTBPs. In some embodiments, antibodies are provided that selectivelybind one or more combinatorial epitopes between GARP and proTGF-β. Insome embodiments of the present invention, compounds and/or compositionsare provided which induce release of TGF-β from GARP-proTGF-β complexes.Such antibodies may be selected for their ability to bind to GARPprodomain binary complexes but not GARP-proTGF-β ternary complexes,GARPs alone, or prodomains alone.

Alternatively or additionally, antibodies of the present invention mayfunction as ligand mimetics which would induce internalization of GPCs.Such antibodies may act as nontraditional payload carriers, acting todeliver and/or ferry bound or conjugated drug payloads to specific GPCand/or GPC-related sites.

Changes elicited by antibodies of the present invention may result inneomorphic changes in the cell. As used herein, the term “neomorphicchange” refers to a change or alteration that is new or different. Forexample, an antibody that elicits the release or stabilization of one ormore growth factor not typically associated with a particular GPCtargeted by the antibody, would be a neomorphic antibody and the releasewould be a neomorphic change.

In some embodiments, compounds and/or compositions of the presentinvention may act to alter and/or control proteolytic events. In someembodiments, such proteolytic events may be intracellular orextracellular. In some embodiments, such proteolytic events may includethe alteration of furin cleavage and/or other proteolytic processingevents. In some embodiments, such proteolytic events may compriseproteolytic processing of growth factor signaling molecules ordownstream cascades initiated by growth factor signaling molecules.

In some embodiments, compounds and/or compositions of the presentinvention may induce or inhibit dimerization or multimerization ofgrowth factors (ligands) or their receptors. In some embodiments, suchactions may be through stabilization of monomeric, dimeric or multimericforms or through the disruption of dimeric or multimeric complexes.

In some embodiments, compounds and/or compositions of the presentinvention may act on homo and/or heterodimers of the monomeric unitscomprising either receptor groups or GPCs or other signaling moleculepairs.

Antibodies of the present invention may be internalized into cells priorto binding target antigens. Upon internalization, such antibodies mayact to increase or decrease one or more signaling events, release orstabilize one or more GPCs, block or facilitate growth factor releaseand/or alter one or more cell niche.

In some embodiments, compounds and/or compositions of the presentinvention may also alter the residence time of one or more growth factorin one or more GPC and/or alter the residence time of one or more GPC inthe extracellular matrix and/or cellular matrix. Such alterations mayresult in irreversible localization and/or transient localization.

Antibodies of the present invention may be designed, manufactured and/orselected using any methods known to one of skill in the art. In someembodiments, antibodies and/or antibody producing cells of the presentinvention are produced according to any of the methods listed in U.S.Provisional Patent Applications 61/722,919, filed Nov. 6, 2012 and61/722,969, filed Nov. 6, 2012, the contents of each of which are hereinincorporated by reference in their entireties.

Antibody Generation in Knockout Mice

In some embodiments, antibodies of the current invention may begenerated in knockout mice that lack a gene encoding one or more desiredantigens. Such mice would not be tolerized to such antigens andtherefore may be able to generate antibodies against them that couldcross react with human and mouse forms of the antigen. For theproduction of monoclonal antibodies, host mice are immunized with thetarget peptide to elicit lymphocytes that specifically bind thatpeptide. Lymphocytes are collected and fused with an immortalized cellline. The resulting hybridoma cells are cultured in a suitable culturemedium with a selection agent to support the growth of only the fusedcells.

In some embodiments, knocking out one or more growth factor gene may belethal and/or produce a fetus or neonate that is non-viable. In someembodiments, neonatal animals may only survive for a matter of weeks(e.g. 1, 2, 3, 4 or 5 weeks). In such embodiments, immunizations may becarried out in neonatal animals shortly after birth. Oida et al (Oida,T. et al., TGF-β induces surface LAP expression on Murine CD4 T cellsindependent of FoxP3 induction. PLOS One. 2010. 5(11):e15523)demonstrate immunization of neonatal TGF-β knockout mice through the useof galectin-1 injections to prolong survival (typically 3-4 weeks afterbirth in these mice). Mice were immunized with cells expressing murineTGF-β every other day for 10 days beginning on the 8^(th) day afterbirth and spleen cells were harvested on day 22 after birth. Harvestedspleen cells were fused with myeloma cells and of the resultinghybridoma cells, many were found to successfully produce anti-LAPantibodies. In some embodiments of the present invention, these methodsmay be used to generate antibodies. In some embodiments, such methodsmay comprise the use of human antigens. In some embodiments, cells usedfor immunization may express TGF-β and GARP. In such embodiments, GARPsmay be expressed with native transmembrane domains to allow forGARP-TGF-β complexes to remain tethered to the cell surface of thetransfected cells used from immunization. Some antigens may compriseproTGF-β1 tethered to LTBP (e.g. LTBP1S.) In some cases, recombinantproteins related to other TGF-β family members may be used as antigens.

Methods of the present invention may also comprise one or more steps ofthe immunization methods described by Oida et al combined with one ormore additional and/or modified steps. Modified steps may include, butare not limited to the use of alternate cell types for fusions, thepooling of varying number of spleen cells when performing fusions,altering the injection regimen, altering the date of spleen cellharvest, altering immunogen and/or altering immunogen dose. Additionalsteps may include the harvesting of other tissues (e.g. lymph nodes)from immunized mice.

Activating and Inhibiting Antibodies

Antibodies of the present invention may comprise activating orinhibiting antibodies. As used herein, the term “activating antibody”refers to an antibody that promotes growth factor activity. Activatingantibodies include antibodies targeting any epitope that promotes growthfactor activity. Such epitopes may lie on prodomains (e.g. LAPs andLAP-like domains) growth factors or other epitopes that when bound byantibody, lead to growth factor activity. Activating antibodies of thepresent invention may include, but are not limited to TGF-β-activatingantibodies, GDF-8-activating antibodies, GDF-11-activating antibodiesand BMP-activating antibodies.

As used herein, the term “inhibiting antibody” refers to an antibodythat reduces growth factor activity. Inhibiting antibodies includeantibodies targeting any epitope that reduces growth factor activitywhen associated with such antibodies. Such epitopes may lie onprodomains (e.g. LAPs and LAP-like domains) growth factors or otherepitopes that lead to reduced growth factor activity when bound byantibody. Inhibiting antibodies of the present invention may include,but are not limited to TGF-β-inhibiting antibodies, GDF-8-inhibitingantibodies, GDF-11-inhibiting antibodies and BMP-inhibiting antibodies.

Embodiments of the present invention include methods of using activatingand/or inhibiting antibodies in solution, in cell culture and/or insubjects to modify growth factor signaling.

Anti-LAP and Anti-LAP-Like Domain Antibodies

In some embodiments, compounds and/or compositions of the presentinvention may comprise one or more antibody targeting a prodomain,including LAP and/or LAP-like domains. Such antibodies may reduce orelevate growth factor signaling depending on the specific LAP orLAP-like domain that is bound and/or depending on the specific epitopetargeted by such antibodies. Anti-LAP and/or anti-LAP-like proteinantibodies of the invention may promote dissociation of free growthfactors from GPCs. Such dissociation may be induced upon antibodybinding to a GPC or dissociation may be promoted by preventing thereassociation of free growth factor with LAP or LAP-like protein. Insome cases, anti-TGF-β LAP antibodies are provided. Anti-TGF-β LAPantibodies may comprise TGF-β-activating antibodies. Such antibodies mayincrease TGF-β activity, in some cases through by releasing TGF-β freegrowth factor from latent GPCs and/or preventing the reassociation offree TGF-β growth factor with LAP. In some cases, anti-TGF-β LAPantibodies may increase TGF-β activity more favorably when proTGF-β isassociated with LTBP. In some cases, anti-TGF-β LAP antibodies mayincrease TGF-β activity more favorably when proTGF-β is associated withGARP. In some cases, anti-TGF-β LAP antibodies may functionsynergistically with other TGF-β activators (e.g. αvβ₆ and/or α_(v)β₈)to increase TGF-β activity.

Variations

Compounds and/or compositions of the present invention may exist as awhole polypeptide, a plurality of polypeptides or fragments ofpolypeptides, which independently may be encoded by one or more nucleicacids, a plurality of nucleic acids, fragments of nucleic acids orvariants of any of the aforementioned. As used herein, the term“polypeptide” refers to a polymer of amino acid residues (natural orunnatural) linked together most often by peptide bonds. The term, asused herein, refers to proteins, polypeptides, and peptides of any size,structure, or function. In some instances the polypeptide encoded issmaller than about 50 amino acids and the polypeptide is then termed apeptide. If the polypeptide is a peptide, it will be at least about 2,3, 4, or at least 5 amino acid residues long. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidesand may be associated or linked. The term polypeptide may also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid.

As used herein, the term “polypeptide variant” refers to molecules whichdiffer in their amino acid sequence from a native or reference sequence.The amino acid sequence variants may possess substitutions, deletions,and/or insertions at certain positions within the amino acid sequence,as compared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” refers to a variant which contains one or moreamino acids which would mimic an activated sequence. For example,glutamate may serve as a mimic for phospho-threonine and/orphospho-serine. Alternatively, variant mimics may result in deactivationor in an inactivated product containing the mimic, e.g., phenylalaninemay act as an inactivating substitution for tyrosine; or alanine may actas an inactivating substitution for serine. The amino acid sequences ofthe compounds and/or compositions of the invention may comprisenaturally occurring amino acids and as such may be considered to beproteins, peptides, polypeptides, or fragments thereof. Alternatively,the compounds and/or compositions may comprise both naturally andnon-naturally occurring amino acids.

As used herein, the term “amino acid sequence variant” refers tomolecules with some differences in their amino acid sequences ascompared to a native or starting sequence. The amino acid sequencevariants may possess substitutions, deletions, and/or insertions atcertain positions within the amino acid sequence. As used herein, theterms “native” or “starting” when referring to sequences are relativeterms referring to an original molecule against which a comparison maybe made. Native or starting sequences should not be confused with wildtype sequences. Native sequences or molecules may represent thewild-type (that sequence found in nature) but do not have to beidentical to the wild-type sequence.

Ordinarily, variants will possess at least about 70% homology to anative sequence, and preferably, they will be at least about 80%, morepreferably at least about 90% homologous to a native sequence.

As used herein, the term “homology” as it applies to amino acidsequences is defined as the percentage of residues in the candidateamino acid sequence that are identical with the residues in the aminoacid sequence of a second sequence after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. It is understood that homology depends on a calculation of percentidentity but may differ in value due to gaps and penalties introduced inthe calculation.

As used herein, the term “homolog” as it applies to amino acid sequencesis meant the corresponding sequence of other species having substantialidentity to a second sequence of a second species.

As used herein, the term “analog” is meant to include polypeptidevariants which differ by one or more amino acid alterations, e.g.,substitutions, additions or deletions of amino acid residues that stillmaintain the properties of the parent polypeptide.

As used herein, the term “derivative” is used synonymously with the term“variant” and refers to a molecule that has been modified or changed inany way relative to a reference molecule or starting molecule.

The present invention contemplates several types of compounds and/orcompositions which are amino acid based including variants andderivatives. These include substitutional, insertional, deletional andcovalent variants and derivatives. As such, included within the scope ofthis invention are compounds and/or compositions comprisingsubstitutions, insertions, additions, deletions and/or covalentmodifications. For example, sequence tags or amino acids, such as one ormore lysines, can be added to peptide sequences of the invention (e.g.,at the N-terminal or C-terminal ends). Sequence tags can be used forpeptide purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to proteins are those that haveat least one amino acid residue in a native or starting sequence removedand a different amino acid inserted in its place at the same position.The substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein, the term “conservative amino acid substitution” refersto the substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

As used herein, the term “insertional variants” when referring toproteins are those with one or more amino acids inserted immediatelyadjacent to an amino acid at a particular position in a native orstarting sequence. As used herein, the term “immediately adjacent”refers to an adjacent amino acid that is connected to either thealpha-carboxy or alpha-amino functional group of a starting or referenceamino acid.

As used herein, the term “deletional variants” when referring toproteins, are those with one or more amino acids in the native orstarting amino acid sequence removed. Ordinarily, deletional variantswill have one or more amino acids deleted in a particular region of themolecule.

As used herein, the term “derivatives,” as referred to herein includesvariants of a native or starting protein comprising one or moremodifications with organic proteinaceous or non-proteinaceousderivatizing agents, and post-translational modifications. Covalentmodifications are traditionally introduced by reacting targeted aminoacid residues of the protein with an organic derivatizing agent that iscapable of reacting with selected side-chains or terminal residues, orby harnessing mechanisms of post-translational modifications thatfunction in selected recombinant host cells. The resultant covalentderivatives are useful in programs directed at identifying residuesimportant for biological activity, for immunoassays, or for thepreparation of anti-protein antibodies for immunoaffinity purificationof the recombinant glycoprotein. Such modifications are within theordinary skill in the art and are performed without undueexperimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the proteins used in accordance withthe present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

Covalent derivatives specifically include fusion molecules in whichproteins of the invention are covalently bonded to a non-proteinaceouspolymer. The non-proteinaceous polymer ordinarily is a hydrophilicsynthetic polymer, i.e. a polymer not otherwise found in nature.However, polymers which exist in nature and are produced by recombinantor in vitro methods are useful, as are polymers which are isolated fromnature. Hydrophilic polyvinyl polymers fall within the scope of thisinvention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularlyuseful are polyvinylalkylene ethers such a polyethylene glycol,polypropylene glycol. The proteins may be linked to variousnon-proteinaceous polymers, such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

As used herein, the term “features” when referring to proteins aredefined as distinct amino acid sequence-based components of a molecule.Features of the proteins of the present invention include surfacemanifestations, local conformational shape, folds, loops, half-loops,domains, half-domains, sites, termini or any combination thereof.

As used herein, the term “surface manifestation” when referring toproteins refers to a polypeptide based component of a protein appearingon an outermost surface.

As used herein, the term “local conformational shape” when referring toproteins refers to a polypeptide based structural manifestation of aprotein which is located within a definable space of the protein.

As used herein, the term “fold”, when referring to proteins, refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein, the term “turn” as it relates to protein conformation,refers to a bend which alters the direction of the backbone of a peptideor polypeptide and may involve one, two, three or more amino acidresidues.

As used herein, the term “loop,” when referring to proteins, refers to astructural feature of a peptide or polypeptide which reverses thedirection of the backbone of a peptide or polypeptide and comprises fouror more amino acid residues. Oliva et al. have identified at least 5classes of protein loops (Oliva, B. et al., An automated classificationof the structure of protein loops. J Mol Biol. 1997. 266(4):814-30.)

As used herein, the term “half-loop,” when referring to proteins, refersto a portion of an identified loop having at least half the number ofamino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein, the term “domain,” when referring to proteins, refers toa motif of a polypeptide having one or more identifiable structural orfunctional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions.)

As used herein, the term “half-domain,” when referring to proteins,refers to a portion of an identified domain having at least half thenumber of amino acid resides as the domain from which it is derived. Itis understood that domains may not always contain an even number ofamino acid residues. Therefore, in those cases where a domain containsor is identified to comprise an odd number of amino acids, a half-domainof the odd-numbered domain will comprise the whole number portion ornext whole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein, the terms “site,” as it pertains to amino acid basedembodiments is used synonymously with “amino acid residue” and “aminoacid side chain”. A site represents a position within a peptide orpolypeptide that may be modified, manipulated, altered, derivatized orvaried within the polypeptide based molecules of the present invention.

As used herein, the terms “termini” or “terminus,” when referring toproteins refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a componentof a molecule of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as site directed mutagenesis. The resulting modifiedmolecules may then be tested for activity using in vitro or in vivoassays such as those described herein or any other suitable screeningassay known in the art.

In some embodiments, compounds and/or compositions of the presentinvention may comprise one or more atoms that are isotopes. As usedherein, the term “isotope” refers to a chemical element that has one ormore additional neutrons. In some embodiments, compounds of the presentinvention may be deuterated. As used herein, the term “deuterate” refersto the process of replacing one or more hydrogen atoms in a substancewith deuterium isotopes. Deuterium isotopes are isotopes of hydrogen.The nucleus of hydrogen contains one proton while deuterium nucleicontain both a proton and a neutron. The compounds and/or compositionsof the present invention may be deuterated in order to change one ormore physical property, such as stability, or to allow compounds and/orcompositions to be used in diagnostic and/or experimental applications.

Conjugates and Combinations

It is contemplated by the present invention that the compounds and/orcompositions of the present invention may be complexed, conjugated orcombined with one or more homologous or heterologous molecules. As usedherein, the term “homologous molecule” refers to a molecule which issimilar in at least one of structure or function relative to a startingmolecule while a “heterologous molecule” is one that differs in at leastone of structure or function relative to a starting molecule. Structuralhomologs are therefore molecules which may be substantially structurallysimilar. In some embodiments, such homologs may be identical. Functionalhomologs are molecules which may be substantially functionally similar.In some embodiments, such homologs may be identical.

Compounds and/or compositions of the present invention may compriseconjugates. Such conjugates of the invention may include naturallyoccurring substances or ligands, such as proteins (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein(HDL), or globulin); carbohydrates (e.g., a dextran, pullulan, chitin,chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids.Conjugates may also be recombinant or synthetic molecules, such assynthetic polymers, e.g., synthetic polyamino acids, an oligonucleotide(e.g. an aptamer). Examples of polyamino acids may include polylysine(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamidecopolymer (HIVIPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamidepolymers, or polyphosphazine. Example of polyamines include:polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

In some embodiments, conjugates may also include targeting groups. Asused herein, the term “targeting group” refers to a functional group ormoiety attached to an agent that facilitates localization of the agentto a desired region, tissue, cell and/or protein. Such targeting groupsmay include, but are not limited to cell or tissue targeting agents orgroups (e.g. lectins, glycoproteins, lipids, proteins, an antibody thatbinds to a specified cell type such as a kidney cell or other celltype). In some embodiments, targeting groups may comprise thyrotropins,melanotropins, lectins, glycoproteins, surfactant protein A, mucincarbohydrates, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine, multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, lipids,cholesterol, steroids, bile acids, folates, vitamin B12, biotin, an RGDpeptide, an RGD peptide mimetic or an aptamer.

In some embodiments, targeting groups may be proteins, e.g.,glycoproteins, or peptides, e.g., molecules having a specific affinityfor a co-ligand, or antibodies e.g., an antibody, that binds to aspecified cell type such as a cancer cell, endothelial cell, or bonecell. Targeting groups may also comprise hormones and/or hormonereceptors.

In some embodiments, targeting groups may be any ligand capable oftargeting specific receptors. Examples include, without limitation,folate, GalNAc, galactose, mannose, mannose-6-phosphate, apatamers,integrin receptor ligands, chemokine receptor ligands, transferrin,biotin, serotonin receptor ligands, PSMA, endothelin, GCPII,somatostatin, LDL, and HDL ligands. In some embodiments, targetinggroups are aptamers. Such aptamers may be unmodified or comprise anycombination of modifications disclosed herein.

In still other embodiments, compounds and/or compositions of the presentinvention may be covalently conjugated to cell penetrating polypeptides.In some embodiments, cell-penetrating peptides may also include signalsequences. In some embodiments, conjugates of the invention may bedesigned to have increased stability, increased cell transfection and/oraltered biodistribution (e.g., targeted to specific tissues or celltypes.)

In some embodiments, conjugating moieties may be added to compoundsand/or compositions of the present invention such that they allow theattachment of detectable labels to targets for clearance. Suchdetectable labels include, but are not limited to biotin labels,ubiquitins, fluorescent molecules, human influenza hemaglutinin (HA),c-myc, histidine (His), flag, glutathione S-transferase (GST), V5 (aparamyxovirus of simian virus 5 epitope), biotin, avidin, streptavidin,horse radish peroxidase (HRP) and digoxigenin.

In some embodiments, compounds of the invention may be conjugated withan antibody Fc domain to create an Fc fusion protein. The formation ofan Fc fusion protein with any of the compounds described herein may becarried out according to any method known in the art, including asdescribed in U.S. Pat. Nos. 5,116,964, 5,541,087 and 8,637,637, thecontents of each of which are herein incorporated by reference in theirentirety. Fc fusion proteins of the invention may comprise a compound ofthe invention linked to the hinge region of an IgG Fc via cysteineresidues in the Fc hinge region. Resulting Fc fusion proteins maycomprise an antibody-like structure, but without C_(H1) domains or lightchains. In some cases, Fc fusion proteins may comprise pharmacokineticprofiles comparable to native antibodies. In some cases, Fc fusionproteins of the invention may comprise extended half-life in circulationand/or altered biological activity.

In some embodiments, compounds and/or compositions of the presentinvention may be combined with one another or other molecules in thetreatment of diseases and/or conditions.

Nucleic Acids

In some embodiments, compounds and/or compositions of the presentinvention may be encoded by nucleic acid molecules. Such nucleic acidmolecules include, without limitation, DNA molecules, RNA molecules,polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids andthe like. In some embodiments, the present invention may comprise cellsprogrammed or generated to express nucleic acid molecules encodingcompounds and/or compositions of the present invention.

Methods of Use

Methods of the present invention include methods of modifying growthfactor activity in one or more biological system. Such methods mayinclude contacting one or more biological system with a compound and/orcomposition of the invention. In some cases, these methods includemodifying the level of free growth factor in a biological system (e.g.in a cell niche or subject.) Compounds and/or compostions according tosuch methods may include, but are not limited to biomolecules,including, but not limited to recombinant proteins, protein complexesand/or antibodies described herein.

In some embodiments, methods of the present invention may be used toinitiate or increase growth factor activity, termed “activating methods”herein. Some such methods may comprise growth factor release from a GPCand/or inhibition of growth factor reassociation into a latent GPC. Insome cases, activating methods may comprise the use of an antibody, arecombinant protein and/or a protein complex. According to someactivating methods, one or more activating antibody is provided. In suchmethods, one or more growth factor may be released or prevented frombeing drawn back into a GPC. In one, non-limiting example, an anti-LAPantibody may be provided that enhances dissociation between a growthfactor and a GPC and/or prevents reformation of a GPC.

Embodiments of the present invention include methods of using anti-LAPand/or anti-LAP-like domain antibodies to modify growth factor activity.In some cases, such methods may include the use of anti-TGF-β-LAPantibodies as TGF-β-activating antibodies. In some cases, methods ofusing and/or testing such antibodies may include any of the methodstaught in Tsang, M. et al. 1995. Cytokine 7(5):389-97, the contents ofwhich are herein incorporated by reference in their entirety.

In some embodiments, methods of the present invention may be used toreduce or eliminate growth factor activity, termed “inhibiting methods”herein. Some such methods may comprise growth factor retention in a GPCand/or promotion of reassociation of growth factor into a latent GPC. Insome cases, inhibiting methods may comprise the use of an antibody

Therapeutics

In some embodiments, compositions and methods of the invention may beused to treat a wide variety of diseases, disorders and/or conditions.In some cases, such diseases, disorders and/or conditions may beTGF-β-related indications. As used herein, the term “TGF-β-relatedindication” refers to any disease, disorder and/or condition related toexpression, activity and/or metabolism of a TGF-β family member proteinor any disease, disorder and/or condition that may benefit frommodulation of the activity and/or levels of one or more TGF-β familymember protein. TGF-β-related indications may include, but are notlimited to, fibrosis, anemia of the aging, cancer (including, but notlimited to colon, renal, breast, malignant melanoma and glioblastoma)facilitation of rapid hematopoiesis following chemotherapy, bonehealing, endothelial proliferation syndromes, asthma and allergy,gastrointestinal disorders, aortic aneurysm, orphan indications (such asMarfan's syndrome and Camurati-Engelmann disease) obesity, diabetes,arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateralsclerosis (ALS) Parkinson's disease, osteoporosis, osteoarthritis,osteopenia, metabolic syndromes, nutritional disorders, organ atrophy,chronic obstructive pulmonary disease (COPD) and anorexia. Additionalindications may include any of those disclosed in US Pub. No.2013/0122007, U.S. Pat. No. 8,415,459 or International Pub. No. WO2011/151432, the contents of each of which are herein incorporated byreference in their entirety.

Efficacy of treatment or amelioration of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Inconnection with the administration of compositions of the presentinvention, “effective against” for example a cancer, indicates thatadministration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as an improvement of symptoms, a cure, a reduction indisease load, reduction in tumor mass or cell numbers, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating theparticular type of cancer.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given composition or formulation of thepresent invention can also be judged using an experimental animal modelfor the given disease as known in the art. When using an experimentalanimal model, efficacy of treatment is evidenced when a statisticallysignificant change is observed.

Therapeutics for Fibrosis

In some embodiments, compounds and/or compositions of the presentinvention may be useful for altering fibrosis. In some embodiments, suchcompounds and/or compositions are antagonists of TGF-β. TGF-β isrecognized as the central orchestrator of the fibrotic response.Antibodies targeting TGF-β decrease fibrosis in numerous preclinicalmodels. Such antibodies and/or antibody-based compounds includeLY2382770 (Eli Lilly, Indianapolis, Ind.). Also included are thosedescribed in U.S. Pat. No. 6,492,497, U.S. Pat. No. 7,151,169 and U.S.Pat. No. 7,723,486 and U.S. publication US2011/0008364, the contents ofeach of which are herein incorporated by reference in their entirety.

Fibrosis is a common sequela of many types of tissue destructivediseases. When new space is created by the disruption of differentiatedcells, progenitors or stem cells that normally occupy a niche in thetissue, the default pathway appears to be the proliferation ofconnective tissue cells, e.g. fibroblasts, to fill in the empty space.This is accompanied by the production of extracellular matrixconstituents including collagens that result in scarring and permanenteffacement of the tissue.

A difficult aspect of fibrosis is its chronicity, which may requirecontinued therapy until the underlying destruction of parenchymal cellsis terminated or the cells are replaced by stem cell pools, or bytransplantation. Fibrosis is thought to be much easier to arrest than toreverse. The TGF-beta family is of central importance in regulating thegrowth of fibroblastic cells and the production of extracellular matrixconstituents including collagen. Integrins α_(v)β₆ and α_(v)β₈ (andpossibly α_(v)β₁) may participate in activation of TGF-beta1 and 3. Theintegrin VLA-1 is a receptor for collagen and is expressed onlymphocytes only late after their activation and is strongly implicatedin the development of fibrotic disease.

In some embodiments, compounds and/or compositions of the presentinvention are designed to block integrin α_(v)β₆, α_(v)β₈ and α_(v)β₁activation of TGF-beta for inhibiting fibrosis. In some embodiments,compounds and/or compositions of the present invention are designed totarget interaction sites between GPCs and LTBPs while leavinginteraction sites between GPCs and GARP unaffected. Such compoundsand/or compositions of the present invention may act as inhibitoryantibodies, preventing growth factor signaling and inhibiting fibrosis.In some embodiments, compounds and/or compositions of the presentinvention are designed to target one or more of TGF-β1, 2 and 3 orchimeric antigens thereof.

Fibrotic indications for which compounds and/or compositions of thepresent invention may be used therapeutically include, but are notlimited to lung indications [e.g. Idiopathic Pulmonary Fibrosis (IPF),Chronic Obstructive Pulmonary Disorder (COPD), Allergic Asthma, AcuteLung injury, Eosinophilic esophagitis, Pulmonary arterial hypertensionand Chemical gas-injury,] kidney indications [e.g. Diabeticglomerulosclerosis, Focal segmental glomeruloclerosis (FSGS), Chronickidney disease, Fibrosis associated with kidney transplantation andchronic rejection, IgA nephropathy and Hemolytic uremic syndrome,] liverfibrosis [e.g. Non-alcoholic steatohepatitis (NASH), Chronic viralhepatitis, Parasitemia, Inborn errors of metabolism, Toxin-mediatedfibrosis, such as alcohol fibrosis, Non-alcoholicsteatohepatitis-hepatocellular carcinoma (NASH-HCC), Primary biliarycirrhosis and Sclerosing cholangitis,] cardiovascular fibrosis (e.g.cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis andrestenosis) systemic sclerosis, skin fibrosis (e.g. Skin fibrosis insystemic sclerosis, Diffuse cutaneous systemic sclerosis, Scleroderma,Pathological skin scarring, Keloid, Post surgical scarring, Scarrevision surgery, Radiation-induced scarring and Chronic wounds) andcancers or secondary fibrosis (e.g. Myelofibrosis, Head and Neck Cancer,M7 acute Megakaryoblastic Leukemia and Mucositis.) Other diseases,disorders or conditions related to fibrosis that may be treated usingcompounds and/or compositions of the present invention, include, but arenot limited to Marfan's Syndrome, Stiff Skin Syndrome, Scleroderma,Rheumatoid arthritis, bone marrow fibrosis, Crohn's disease, Ulcerativecolitis, Systemic lupus erythematosus, Muscular Dystrophy, Dupuytren'scontracture, Camurati-Engelmann Disease, Neural scarring, Proliferativevitreoretinopathy, corneal injury, complications after glaucoma drainagesurgery and Multiple Sclerosis.

Assays useful in determining the efficacy of the compounds and/orcompositions of the present invention for the alteration of fibrosisinclude, but are not limited to, histological assays for countingfibroblasts and basic immunohistochemical analyses known in the art.

Animal models are also available for analysis of the efficacy ofcompounds and/or compositions of the present invention in alteringfibrosis. Examples of animal fibrosis models useful for such analysismay include, for example, any of those taught by Schaefer, D. W. et al.,2011. Eur Respir Rev. 20: 120, 85-97, the contents of which are hereinincorporated by reference in their entirety. Such models may include,but are not limited to those described in Table 1 of that publication,including lung models, renal models, liver models, cardiovascular modelsand/or collagen-induced models. Schaefer et al also teach the use ofpirfenidone in the treatment of fibrosis. In some cases, compoundsand/or compositions of the present invention may be used in combinationwith pirfenidone.

In some cases, compounds and/or composition of the invention may be usedin the treatment of lung fibrosis. Lung fibrosis models may be used inthe development and/or testing of compounds and/or compositions of theinvention. Lung fibrosis models may include the bleomycin induced lunginjury models and/or chronic bleomycin induced lung injury models.Bleomycin induced lung injury models may be carried out as described bySchaefer et al, and also by Horan et al. (Horan G. S. et al., 2008. Am JRespir Crit Care Med, 177(1):56-65. Epub 2007 Oct. 4, the contents ofeach of which are herein incorporated by reference in their entirety.)According to the Horan study, SV129 mice are tracheally exposed tobleomycin which results in the development of lung fibrosis. With thismodel, potential therapeutics are administered through intraperitonealinjections while postmortem lung tissue or bronchoalveolar lavagecollections can be assayed for levels of hydroxyproline as an indicatorof fibrotic activity. Using the same technique, mice carrying aluciferase reporter gene, driven by the collagen Iα2 gene promoter maybe used in the model so that fibrotic activity may be determined byluciferase activity assay as a function of collagen gene induction.Additional bleomycin induced lung models may be carried out according tothose described by Thrall et al (Thrall, R. S. et al., 1979. Am JPathol. 95:117-30, the contents of which are herein incorporated byreference in their entirety.) Additional lung models may include themouse asthma models. Airway remodeling (lung fibrosis) may be a seriousproblem in subjects with chronic asthma. Asthma models may include anyof those described by Nials et al (Nials, A. T. et al., 2008. DiseaseModels and Mechanisms. 1:213-20, the contents of wich are hereinincorporated by reference in their entirety.) Models of chronicobstructive pulmonary disease (COPD) may be used. Such models mayinclude any of those described by Vlahos et al (Vlahos, R. et al., 2014.Clin Sci. 126:253-65, the contents of which are herein incorporated byreference in their entirety.) Models of cigarette smoking emphysema maybe used. Such models may be carried out as described in Ma et al. 2005.J Clin Invest. 115:3460-72, the contents of which are hereinincorporated by reference in their entirety. Models of chronic pulmonaryfibrosis may be used. Such models in rodents may be carried outaccording to the intratracheal fluorescein isothiocyanate (FITC)instillation model described in Roberts, S. N. et al. 1995. J Pathol.176(3):309-18, the contents of which are herein incorporated byreference in their entirety. Models of asbestos and silica induced lunginjury may also be used. Such models may be carried out as described inCoin, P. G. et al., 1996. Am J Respir Crit Care Med. 154(5):1511-9, thecontents of which are herein incorporated by reference in theirentirety. In some cases, models of lung irradiation may be used. Suchmodels may be carried out as described in Pauluhn, J. et al. 2001.Toxicology. 161:153-63, the contents of which are herein incorporated byreference in their entirety. In some cases, phorbol myristate acetate(PMA)-induced lung injury models may be used. Such models may be carriedout as described in Taylor, R. G. et al., 1985. Lab Invest. 52(1):61-70,the contents of which are herein incorporated by reference in theirentirety.

Renal fibrosis models may be utilized to develop and/or test compoundsand/or compositions of the present invention. In some embodiments, awell established model of renal fibrosis, unilateral ureteralobstruction (UUO) model, may be used. In this model, mice are subjectedto proximal ureteral ligation. After a period of hours to days, fibrosisis examined in the regions blocked by ligation (Ma, L. J. et al., 2003.American Journal of Pathology. 163(4):1261-73, the contents of which areherein incorporated by reference in their entirety.) In one example,this method was utilized by Meng, X. M. et al. (Meng, X. M. et al.,Smad2 Protects against TGF-beta/Smad3-Mediated Renal Fibrosis. J Am SocNephrol. 2010 September; 21(9):1477-87. Epub 2010 Jul. 1) to examine therole of SMAD-2 in renal fibrosis. SMAD-2 is an intracellular member ofthe TGF-beta cell signaling pathway. In some cases, cyclosporineA-induced nephropathy models may be used. Such models may be carried outas described in Ling, H. et al., 2003. J Am Soc Nephrol. 14:377-88, thecontents of which are herein incorporated by reference in theirentirety. In some cases, renal models of Alport Syndrome may be used.Transgenic mice with collagen III knockout may be used in Alportsyndrome studies. These mice develop progressive fibrosis in theirkidneys. Alport syndrome models may be carried out as described inKoepke, M. L. et al., 2007. Nephrol Dial Transplant. 22(4):1062-9 and/orHahm, K. et al., 2007. Am J Pathol. 170(1):110-5, the contents of eachof which are herein incorporated by reference in their entirety.

In some cases, models of cardiovascular fibrosis may be used to developand/or test compounds and/or compositions of the invention for treatmentof cardiovascular fibrotic indications. In some cases, vascular injurymodels may be used. Such models may include balloon injury models. Insome cases, these may be carried out as described in Smith et al., 1999.Circ Res. 84(10):1212-22, the contents of which are herein incorporatedby reference in their entirety. Blocking TGF-β in this model was shownto block neointima formation. Accordingly, TGF-β inhibiting antibodiesof the present invention may be used to reduce and/or block neointimaformation.

In some embodiments, models of liver fibrosis may be used to developand/or test compounds and/or compositions of the invention for treatmentof liver fibrotic indications. Liver models may include any of thosedescribed in Iredale, J. P. 2007. J Clin Invest. 117(3):539-48, thecontents of which are herein incorporated by reference in theirentirety. These include, but are not limited to, any of the modelslisted in Tables 1 and/or 2. In some cases, liver models may includecarbon tetrachloride induced liver fibrosis models. Such models may becarried out according to the methods described in Fujii, T. et al.,2010. BMC Gastroenterology. 10:79, the contents of which are hereinincorporated by reference in their entirety.

In some embodiments, models of wound healing may be used to developand/or test compounds and/or compositions of the invention for treatmentof fibrotic wound indications. Wound models may include chronic woundmodels.

In some cases, models of GI injury-related fibrosis may be used todevelop and/or test compounds and/or compositions of the invention fortreatment of GI-related fibrosis. Such injury models may include, butare not limited to 2,4,6-trinitrobenzenesulfonic acid (TNBS) inducedcolitis models. Such models may be carried out as described inScheiffele, F. et al., 2002. Curr Protoc Immunol. Chapter 15:Unit 15.19,the contents of which are herein incorporated by reference in theirentirety.

In some embodiments, compounds and/or compositions of the invention maybe used to treat diseases, disorders and/or conditions related to bonemarrow fibrosis. In some cases, bone marrow fibrosis models may be usedto develop and/or test such compounds and/or compositions. Models mayinclude the marrow cell adoptive transfer model described in Lacout, C.et al., 2006. Blood. 108(5):1652-60 and transgenic mouse models,including, but not limited to the model described in Vannucchi, A. M. etal., 2002. Blood. 100(4):1123-32, the contents of each of which areherein incorporated by reference in their entirety. Further models mayinclude models of thrombopoietin-induced myelofibrosis. Such models maybe carried out as described in Chagraoui, H. et al., 2002. Blood.100(10):3495-503, the contents of which are herein incorporated byreference in their entirety.

In some embodiments, compounds and/or compositions of the invention maybe used to treat diseases, disorders and/or conditions related tomuscular dystrophy (MD) including, but not limited to Duchenne MD andBecker MD. In some cases MD models may be used to develop and/or testsuch compounds and/or compositions. Such models may include thosedescribed in Ceco, E. et al., 2013. FEBS J. 280(17):4198-209, thecontents of which are herein incorporated by reference in theirentirety.

Compounds and/or compositions of the invention may, in some cases, becombined with one or more other therapeutics for the treatment of one ormore fibrotic indication. Examples of such other therapeutics mayinclude, but are not limited to LPA1 receptor antagonists, lysyl oxidase2 inhibitors, hedgehog inhibitors, IL-3/IL-4 inhibitors, CTGFinhibitors, anti-α_(v)β₆ antibodies and anti-IL-13 antibodies.

In some cases, compounds and/or compositions of the present inventionare designed to increase TGF-β growth factor activity to promotefibrosis to treat diseases, disorders and/or conditions where fibrosismay be advantageous. Such compounds may include activating antibodies.

Therapeutics for Myelofibrosis

Myelofibrosis is a chronic blood cancer caused by mutations in bonemarrow stem cells. Disease is characterized by an impaired ability tomake normal blood cells. Patients develop splenomegaly and hepatomegalyand excessive fibrosis occurs in the bone marrow. Myeloproliferativeneoplasms (MPNs) are the collective name for three related types ofmyelofibrosis with different clinical features: primary myelofibrosis(PMF), essential thrombocythemia and polycythemia vera. All three haveoveractive signaling of the JAK-STAT cell signaling pathway (Klampfi, etal., 2013. NEJM 369:2379-90, the contents of which are hereinincorporated by reference in their entirety.) Primary myelofibrosis(PMF) is characterized by increased angiogenesis, reticulin and collagenfibrosis. As the disease advances, the number of osteoclasts increaseand bone marrow becomes unaspirable. Some fibrosis of PMF may bereversed by stem cell transplantation (SCT.) 98% of individuals withpolycythemia vera have mutated JAK2 leading to overactive JAK-STATsignaling.

Current therapeutics for MPNs include allogeneic hematopoietic celltransplantation (HCT) and Janus kinase (JAK) inhibition. Allogeneic HCTis associated with up to 10% mortality as well as graft failure andsignificant side effects and toxicity. JAK inhibition therapy comprisesthe use of Ruxolitinib (Rux) a small molecule inhibitor of JAK2 that wasapproved in 2011 to treat MPNs. Rux is marketed under the names JAKAFI®and JAKAVI® by Incyte pharmaceuticals (Wilmington, Del.) and Novartis(Basel, Switzerland). Although able to improve splenomegaly andhepatomegaly, Rux is not curative and some studies do not show muchbenefit (Odenike, O., 2013. Hematology. 2013(1):545-52, the contents ofwhich are herein incorporated by reference in their entirety.)

In some cases, compounds and/or compositions of the invention may beused to treat myeloproliferative disorders, including, but not limitedto primary myelofibrosis, secondary myelofibrosis, essentialthrombocythemia, polycythemia vera, idiopathic myelofibrosis and chronicmyeloid leukemia. In some cases, treatments may be carried out incombination with one or more known therapies for myelofibrosis,including, but not limited to allogeneic HCT, JAK inhibition,fresolimumab (GC1008; Genzyme, Cambridge, Mass.) treatment to blockTGF-β1, 2 and 3 (Mascarenhas, J. et al., 2014. Leukemia and Lymphoma.55:450-2, the contents of which are herein incorporated by reference intheir entirety) simtuzumab (Gilead Biosciences, Foster City, Calif.)treatment to block lysyl oxidase activity and collagen cross-linking andPentraxin-2 (Promedior, Lexington, Mass.) treatment to stimulateregulatory macrophages and inhibit myelofibroblasts. In some cases,models of myeloproliferative disorders may be used to develop and/ortest such compounds and/or compositions of the invention intended forthe treatment of myelofibrosis. Models may include the marrow celladoptive transfer model described in Lacout, C. et al., 2006. Blood.108(5):1652-60 and transgenic mouse models, including, but not limitedto the model described in Vannucchi, A. M. et al., 2002. Blood.100(4):1123-32, the contents of each of which are herein incorporated byreference in their entirety. Myelofibrosis models may includethrombopoietin-induced myelofibrosis. Such models may be carried out asdescribed in Chagraoui, H. et al., 2002. Blood. 100(10):3495-503, thecontents of which are herein incorporated by reference in theirentirety. TGF-β1 has been shown to be the primary agonist of fibrosisaccording to this model. Further myelofibrosis models may be carried outas described in Mullally, A. et al., 2010. Cancer Cell. 17:584-96, thecontents of which are herein incorporated by reference in theirentirety.

Therapeutics for Scarring and Wound Healing

In some embodiments, compounds and/or compositions of the presentinvention may be useful in altering wound healing and/or scar formation.In some cases, compounds and/or compositions of the invention may ensureproper wound healing (including, but not limited to chronic wounds.) Insome cases, compounds and/or compositions of the invention may be usedfor reducing, treating and or preventing scar formation. Such compoundsand/or compositions may comprise anti-TGF-β antibodies. In some cases,TGF-β-activating antibodies may be used to promote healing in wounds.

Therapeutics for Disorders of Iron Metabolism

In some embodiments, methods, compounds and/or compositions of thepresent invention may be used to treat disorders of iron metabolism.Such disorders may include disorders comprising reduced iron levels(e.g. anemias) or disorders comprising elevated iron levels (e.g.hemochromatosis.) BMP-6 and hemojuvelin interact to modulate hepcidinexpression. Some methods, compounds and/or compositions of disclosedherein may be used to alter hepcidin levels, thereby regulating bodilyiron levels.

Some embodiments of the present invention may comprise hepcidin agonistsor hepcidin antagonists. Hepcidin agonists may activate or promote theexpression and/or physiological action of hepcidin. Such agonists may beuseful in the treatment or prevention of iron overload due to lowhepcidin levels and/or activity. In some cases, agonists may not reverseestablished iron overload, but may diminish iron damage to tissues. Somehepcidin agonists of the present invention may elevate production ofhepcidin through activating and/or enhancing BMP-6/hemojuvelinsignaling.

Hepcidin antagonists may block or reduce the expression and/orphysiological action of hepcidin. Such antagonists may be useful in thecase of iron deficiency due to high hepcidin levels. In someembodiments, hepcidin antagonists of the present invention may compriseantibodies that disrupt BMP-6 signaling through hemojuvelin.

Anemias are conditions and/or diseases associated with decreased numbersof red blood cells and/or hemoglobin. Compounds and/or compositions ofthe present invention may be useful in treating anemias. Such anemiasmay include anemia of chronic disease (ACD), which is also referred toas anemia of inflammation (AI). Subjects with ACD, may suffer fromchronic renal failure or acute inflammation due to rheumatoid arthritis,cancer, infection, etc. Subjects suffering from ACD typically compriseelevated levels of hepcidin and impaired erythropoiesis. In a study bySasu et al (Sasu et al., 2010. Blood. 115(17):3616-24) an antibody withhigh affinity for hepcidin was effective in treating murine anemia in amouse model of inflammation. The studies found that the most effectivetreatments involved combining the antibody with anerythropoiesis-stimulating agent (ESA.) Accordingly, some compoundsand/or compositions of the present invention may be used in combinationwith ESAs to increase efficacy. Current anti-hepcidin antibodies beingtested for treatment of ACD include Ab12B9 (Amgen, Thousand Oaks,Calif.) and LY2787106 (Eli Lilly, Indianapolis, Ind.) FG4592 (FibroGen,San Francisco, Calif.) is a small molecule inhibitor ofhypoxia-inducible factor (HIF) that is also currently used to treatanemia.

In some cases, compounds and/or compositions of the present inventionmay be used to treat subjects with iron deficiency anemia (IDA)associated with gastric bypass surgery and/or inflammatory bowel disease(IBD.) Gastric bypass surgery leaves subjects with a reduced ability tometabolize iron due to bypass of the proximal gastric pouch and duodenum(Warsh et al., 2013, the contents of which are herein incorporated byreference in their entirety.) IBD patients often suffer from irondeficiency due to intestinal blood loss and decreased absorption due toinflammation.

Some compounds and/or compositions of the present invention may be usedto treat subjects suffering from iron-refractory iron deficiency anemia(IRIDA.) IRIDA is a genetic disease caused by a defect in the enzymeMatriptase-2 (De Falco, L. et al., 2013, the contents of which areherein incorporated by reference in their entirety.) Matriptase-2, atransmembrane serine protease, is an important hepcidin regulator.Matriptase-2 is capable of enzymatic cleavage of hemojuvelin. Subjectswith defective Matriptase-2 activity have elevated levels ofhemojuvelin, due to lack of degradation, and therefore hepcidinexpression remains high and iron levels are reduced. Characteristics ofthe disease include, but are not limited to microcytic hypochromicanemia, low saturation of transferrin and normal to high levels ofhepcidin. Some subjects with IRIDA are diagnosed soon after birth, butmany are not diagnosed until adulthood. Treatments described herein maybe used to modulate irregular hepcidin levels associated with IRIDA.

Iron overloading anemias can occur as a result of blood transfusion.Excess iron associated with transfused blood cannot be secretednaturally and requires additional treatments for removal, such aschelation therapy. Such therapy is generally not well tolerated and maycomprise many side effects. Thus, there is a clinical need for new,better tolerated therapies. Additional therapies include EXJADE®, forthe treatment of patients, age 10 and older, withnon-transfusion-dependent thalassemia (NTDT) syndromes. Also included isACE-536, a ligand trap that blocks TGF-β superfamily members. BothEXJADE and ACE-536 are known to elevate erythropoiesis. In someembodiments, compounds and/or compositions of the present invention maybe used to control iron overloading. Some such embodiments may functionto redistribute iron from parenchyma to macrophages where iron is bettertolerated. In some cases this may be carried out through elevation ofhepcidin levels. In studies by Gardenghi et al (Gardenghi et al., 2010,JCI. 120(12):4466-77) overexpression of murine hepcidin was able toincrease hemoglobin levels and decrease iron overload in mouse model ofβ-thalassemia and a mouse model of hemochromatosis (Viatte et al., 2006,Blood. 107:2952.)

GDF-15 levels in circulation have been found to negatively correlatewith hepcidin levels, suggesting a role for GDF-15 in iron loadingand/or metabolism (Finkenstedt et al., 2008. British Journal ofHaematology. 144:789-93, the contents of which are herein incorporatedby reference in their entirety.) Transcription of the gene encodingGDF-15 may be upregulated under stress and/or hypoxic conditions. Insome cases, compounds and/or compositions of the present invention maybe used to treat subjects suffering from iron disorders and/or anemiasby altering GDF-15 signaling activity. Such compounds and/orcompositions may comprise antibodies capable of stabilizing ordestabilizing the GDF-15 GPC or through modulation of one or moreinteraction between GDF-15 and one or more co-factor.

Hemochromatosis is a disease characterized by iron overload due tohyperabsorption of dietary iron. In hereditary hemochromatosis (HH) thisoverload is caused by inheritance of a common autosomal recessive copyof the HFE gene from both parents. In such cases, iron may be overloadedin plasma as well as in organs and tissues, including, but not limitedto the pancreas, liver and skin, leading to damage caused by irondeposits (Tussing-Humphreys et al, 2013.) Current therapies for HH mayinclude phlebotomy, multiple times per year. In some embodiments,compounds and/or compositions of the present invention may be used totreat HH by modulating subject iron levels.

Mutations in the hepcidin (HAMP) and/or hemojuvelin (HFE2) genes areresponsible for a severe form of hemochromatosis known as juvenilehemochromatosis (Roetto et al., 2003; Papanikolauou et al., 2004.) Somemutations of hemojuvelin associated with juvenile hemochromatosis leadto protein misfolding and reduce hemojuvelin secretion from the cell,thus decreasing overall hemojuvelin signaling activity. Other mutationsaffect hemojuvelin interactions with other signaling molecules.Hemojuvelin comprising the mutation G99R, for example, is unable to bindBMP-2. Hemojuvelin comprising the mutation L101P is unable to associatewith either BMP-2 or neogenin. Some therapeutic embodiments of thepresent invention may comprise the modulation of hemojuvelin signaling.

During chemotherapy, cell division is temporarily halted to prevent thegrowth and spread of cancerous cells. An unfortunate side effect is theloss of red blood cells which depend on active cell division of bonemarrow cells. In some embodiments, compounds and/or compositions of thepresent invention may be used to treat anemia associated chemotherapy.

In some cases, compounds and/or compositions of the present inventionmay be combined with any of the therapeutics described herein toincrease efficacy.

Therapeutics for Anemia, Thrombocytopenia and Neutropenia

During chemotherapy, cell division is temporarily halted to prevent thegrowth and spread of cancerous cells. An unfortunate side effect is theloss of red blood cells, platelets and white blood cells which depend onactive cell division of bone marrow cells. In some embodiments,compounds and/or compositions of the present invention may be designedto treat patients suffering from anemia (the loss of red blood cells),thrombocytopenia (a decrease in the number of platelets) and/orneutropenia (a decrease in the number of neutrophils).

Therapeutics for Cancer

Various cancers may be treated with compounds and/or compositions of thepresent invention. As used herein, the term “cancer” refers to any ofvarious malignant neoplasms characterized by the proliferation ofanaplastic cells that tend to invade surrounding tissue and metastasizeto new body sites and also refers to the pathological conditioncharacterized by such malignant neoplastic growths. Cancers may betumors or hematological malignancies, and include but are not limitedto, all types of lymphomas/leukemias, carcinomas and sarcomas, such asthose cancers or tumors found in the anus, bladder, bile duct, bone,brain, breast, cervix, colon/rectum, endometrium, esophagus, eye,gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum(chest), mouth, ovaries, pancreas, penis, prostate, skin, smallintestine, stomach, spinal marrow, tailbone, testicles, thyroid anduterus.

In cancer, TGF-β may be either growth promoting or growth inhibitory. Asan example, in pancreatic cancers, SMAD4 wild type tumors may experienceinhibited growth in response to TGF-β, but as the disease progresses,constitutively activated type II receptor is typically present.Additionally, there are SMAD4-null pancreatic cancers. In someembodiments, compounds and/or compositions of the present invention aredesigned to selectively target components of TGF-β signaling pathwaysthat function uniquely in one or more forms of cancer. Leukemias, orcancers of the blood or bone marrow that are characterized by anabnormal proliferation of white blood cells i.e., leukocytes, can bedivided into four major classifications including Acute lymphoblasticleukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenousleukemia or acute myeloid leukemia (AML) (AML with translocationsbetween chromosome 10 and 11 [t(10, 11)], chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15; 17)], and inversions in chromosome 16[inv(16)]; AML with multilineage dysplasia, which includes patients whohave had a prior myelodysplastic syndrome (MDS) or myeloproliferativedisease that transforms into AML; AML and myelodysplastic syndrome(MDS), therapy-related, which category includes patients who have hadprior chemotherapy and/or radiation and subsequently develop AML or MDS;d) AML not otherwise categorized, which includes subtypes of AML that donot fall into the above categories; and e) Acute leukemias of ambiguouslineage, which occur when the leukemic cells cannot be classified aseither myeloid or lymphoid cells, or where both types of cells arepresent); and Chronic myelogenous leukemia (CML).

The types of carcinomas include, but are not limited to,papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma,adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma,rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma,lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, largecell undifferentiated carcinomas, basal cell carcinoma and sinonasalundifferentiated carcinoma.

The types of sarcomas include, but are not limited to, soft tissuesarcoma such as alveolar soft part sarcoma, angiosarcoma,dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor,extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, andAskin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor),malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, andchondrosarcoma.

In some embodiments, compositions and methods of the invention may beused to treat one or more types of cancer or cancer-related conditionsthat may include, but are not limited to colon cancer, renal cancer,breast cancer, malignant melanoma and glioblastomas (Schlingensiepen etal., 2008; Ouhtit et al., 2013.)

High-grade gliomas (e.g. anaplastic astrocytomas and glioblastomas) makeup around 60% of malignant brain tumors. TGF-β2 has been found to beoverexpressed in over 90% of such gliomas and expression levelscorrelate with tumor progression. Further, studies using TGF-02reduction at the mRNA level in cancer patients showed significantimprovement in tumor outcome (Bogdahn et al., 2010.) In light of thesestudies, some compositions of the present invention may be usedtherapeutically to treat individuals with high-grade gliomas. Suchcompositions may act to lower the levels of free TGF-β2 and/or thelevels of TGF-β2 activity.

In some cases, TGF-β2 activity may contribute to tumor developmentthrough modulation of metastasis, angiogenesis, proliferation and/orimmunosuppressive functions that impair immunological tumor surveillance(Schlingensiepen et al., 2008.) A study by Reed et al (Reed et al.,1994) demonstrated TGF-β2 mRNA expression in a large percentage ofmelanocytic lesions including primary invasive melanomas and metastaticmelanomas. Some compounds and/or compositions of the present inventionmay be used to modulate TGF-β2 activity and/or levels in such lesionsand or prevent lesion formation. Melanoma cell growth in the brainparenchyma has also been shown to be influenced by TGF-β2 activity(Zhang et al., 2009.) Some compounds and/or compositions of the presentinvention may be used to prevent or control such cell growth throughmodulation of TGF-β2 activity and/or levels.

Among females worldwide, breast cancer is the most prevalent form ofcancer. Breast cancer metastasis is mediated in part throughinteractions between cancer cells and extracellular matrix components,such as hyaluronic acid (HA.) CD44 has been shown to be the majorreceptor for HA on cancer cells (Ouhtit et al., 2013.) The interactionbetween CD44 and HA leads to modulation of cell motility, survivaladhesion and proliferation. TGF-β2 transcription is also upregulated byCD44 signaling activity and is believe to contribute to resultingchanges in cell motility. Unfortunately, current therapies have limitedefficacy and many carry adverse effects due to a lack of specificity. Insome cases, compounds and/or compositions of the present invention maybe used to alter cellular activities induced by TGF-β2 upregulation.

The invention further relates to the use of compounds and/orcompositions of the present invention for treating one or more forms ofcancer, in combination with other pharmaceuticals and/or othertherapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating these disorders. For example, the compounds and/orcompositions of the present invention can also be administered inconjunction with one or more additional anti-cancer treatments, such asbiological, chemotherapy and radiotherapy. Accordingly, a treatment caninclude, for example, imatinib (Gleevac), all-trans-retinoic acid, amonoclonal antibody treatment (gemtuzumab, ozogamicin), chemotherapy(for example, chlorambucil, prednisone, prednisolone, vincristine,cytarabine, clofarabine, farnesyl transferase inhibitors, decitabine,inhibitors of MDR1), rituximab, interferon-α, anthracycline drugs (suchas daunorubicin or idarubicin), L-asparaginase, doxorubicin,cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide,pentostatin, or cladribine), bone marrow transplant, stem celltransplant, radiation therapy, anti-metabolite drugs (methotrexate and6-mercaptopurine), or any combination thereof.

Radiation therapy (also called radiotherapy, X-ray therapy, orirradiation) is the use of ionizing radiation to kill cancer cells andshrink tumors. Radiation therapy can be administered externally viaexternal beam radiotherapy (EBRT) or internally via brachytherapy. Theeffects of radiation therapy are localized and confined to the regionbeing treated. Radiation therapy may be used to treat almost every typeof solid tumor, including cancers of the brain, breast, cervix, larynx,lung, pancreas, prostate, skin, stomach, uterus, or soft tissuesarcomas. Radiation is also used to treat leukemia and lymphoma.

Chemotherapy is the treatment of cancer with drugs that can destroycancer cells. In current usage, the term “chemotherapy” usually refersto cytotoxic drugs which affect rapidly dividing cells in general, incontrast with targeted therapy. Chemotherapy drugs interfere with celldivision in various possible ways, e.g. with the duplication of DNA orthe separation of newly formed chromosomes. Most forms of chemotherapytarget all rapidly dividing cells and are not specific to cancer cells,although some degree of specificity may come from the inability of manycancer cells to repair DNA damage, while normal cells generally can.

Most chemotherapy regimens are given in combination. Exemplarychemotherapeutic agents include, but are not limited to, 5-FU Enhancer,9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide,Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine,Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors,Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl,D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel,Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR,Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide(VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol,Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline,Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide),Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2,Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate(LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gammalinolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat,Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, MeglamineGLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxalbis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone,Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698,OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARPInhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412,Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS FarnesylTransferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU),Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide,Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase,UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesinesulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD9331.

Biological therapies use the body's immune system, either directly orindirectly, to fight cancer or to lessen the side effects that may becaused by some cancer treatments. In some embodiments, compounds and/orcompositions of the present invention may be considered biologicaltherapies in that they may stimulate immune system action against one ormore tumor, for example. However, this approach may also be consideredwith other such biological approaches, e.g., immune response modifyingtherapies such as the administration of interferons, interleukins,colony-stimulating factors, other monoclonal antibodies, vaccines, genetherapy, and nonspecific immunomodulating agents are also envisioned asanti-cancer therapies to be combined with the compounds and/orcompositions of the present invention.

Small molecule targeted therapy drugs are generally inhibitors ofenzymatic domains on mutated, overexpressed, or otherwise criticalproteins within the cancer cell, such as tyrosine kinase inhibitorsimatinib (Gleevec/Glivec) and gefitinib (Iressa). Examples of monoclonalantibody therapies that can be used with compounds and/or compositionsof the present invention include, but are not limited to, theanti-HER2/neu antibody trastuzumab (Herceptin) used in breast cancer,and the anti-CD20 antibody rituximab, used in a variety of B-cellmalignancies. The growth of some cancers can be inhibited by providingor blocking certain hormones. Common examples of hormone-sensitivetumors include certain types of breast and prostate cancers. Removing orblocking estrogen or testosterone is often an important additionaltreatment. In certain cancers, administration of hormone agonists, suchas progestogens may be therapeutically beneficial.

Cancer immunotherapy refers to a diverse set of therapeutic strategiesdesigned to induce the patient's own immune system to fight the tumor,and include, but are not limited to, intravesical BCG immunotherapy forsuperficial bladder cancer, vaccines to generate specific immuneresponses, such as for malignant melanoma and renal cell carcinoma, andthe use of Sipuleucel-T for prostate cancer, in which dendritic cellsfrom the patient are loaded with prostatic acid phosphatase peptides toinduce a specific immune response against prostate-derived cells.

In some embodiments, compounds and/or compositions of the presentinvention are designed to prevent T cell inhibition. Such compoundsand/or compositions may prevent the dissociation of growth factors fromthe prodomain of the GPC or from extracellular matrix and/or cellularmatrix components including, but not limited to GARPs, fibrillins orLTBPs.

Therapeutics for Bone Healing

Compounds and/or compositions of the present invention may be used totreat bone disorders and/or improve bone healing or repair. Cellularremodeling of bone is a lifelong process that helps to maintain skeletalintegrity. This process involves cycles of osteoclastic bone resorptionand new bone formation that function to repair defects and areas ofweakness in bone. TGF-beta family members, preferably BMPs, are thoughtto be important factors in coupling the processes of resorption andformation by osteoclasts. TGF-beta family members are prevalent in thebone matrix and upregulated by bone injury. TGF-beta family members arealso believed to impart strength to the fully formed bone matrix,imparting resistance to fracture. The role of TGF-beta family members inbone remodeling makes them attractive targets for potential therapeuticsto treat bone disorder and disease.

Numerous diseases and/or disorders affect bones and joints. Suchdiseases and/or disorders may be congenital, genetic and/or acquired.Such diseases and/or disorders include, but are not limited to, bonecysts, infectious arthritis, Paget's disease of the bone,Osgood-Schlatter disease, Kohler's bone disease, bone spurs(osteophytes), bone tumors, craniosynostosis, fibrodysplasia ossificansprogressive, fibrous dysplasia, giant cell tumor of bone,hypophosphatasia, Klippel-Feil syndrome, metabolic bone disease,osteoarthritis, osteitis deformans, osteitis fibrosa cystica, osteitispubis, condensing osteitis, osteitis condensans osteochondritisdissecans, osteochondroma, osteogenesis imperfecta, osteomalacia,osteomyelitis, osteopenia, osteopetrosis, osteoporosis, osteosarcoma,porotic hyperostosis, primary hyperparathyroidism, renal osteodystrophyand water on the knee.

Mouse models for evaluating the effectiveness of therapeutics on bonedevelopment and repair are well known in the art. In one such modeldemonstrated by Mohammad, et al. (Mohammad, K. S. et al., Pharmacologicinhibition of the TGF-beta type I receptor kinase has anabolic andanti-catabolic effects on bone. PLoS One. 2009; 4(4):e5275. Epub 2008Apr. 16), inhibition of the TGF-beta type I receptor was carried out inC57B1/6 mice through twice daily administration of a potent inhibitor,SD-208, by gavage. Subsequently, bone mineral density (BMD) was analyzedusing a PIXImus mouse densitometer (GE Lunar II, Faxitron Corp.,Wheeling, Ill.). Changes in BMD are expressed as a percentage change inthe area scanned. The study found that after 6 weeks of treatment, malemice exhibited a 4.12% increase in bone accrual while female miceexhibited a 5.2% increase.

Compounds and/or compositions of the present invention may be useful astherapies for simple or complex bone fractures and/or bone repair. Insuch treatments, compounds and/or compositions of the present inventionmay be introduced to the site of injury directly or through theincorporation into implantation devices and coated biomatrices.Additionally, treatments are contemplated in which compounds and/orcompositions of the present invention are supplied together with one ormore GPC in a treatment area, facilitating the slow release of one ormore growth factors from such GPCs.

Therapeutics for Angiogenic and Endothelial Proliferation Conditions

The compounds and/or compositions of the present invention may be usedto treat angiogenic and endothelial proliferation syndromes, diseases ordisorders. The term “angiogenesis”, as used herein refers to theformation and/or reorganization of new blood vessels. Angiogenic diseaseinvolves the loss of control over angiogenesis in the body. In suchcases, blood vessel growth, formation or reorganization may beoveractive (including during tumor growth and cancer where uncontrolledcell growth requires increased blood supply) or insufficient to sustainhealthy tissues. Such conditions may include, but are not limited toangiomas, angiosarcomas, telangiectasia, lymphangioma, congenitalvascular anomalies, tumor angiogenesis and vascular structures aftersurgery. Excessive angiogenesis is noted in cancer, maculardegeneration, diabetic blindness, rheumatoid arthritis, psoriasis aswell as many other conditions. Excessive angiogenesis is often promotedby excessive angiogenic growth factor expression. Compounds and/orcompositions of the present invention may act to block growth factorsinvolved in excessive angiogenesis. Alternatively, compounds and/orcompositions of the present invention may be utilized to promote growthfactor signaling to enhance angiogenesis in conditions whereangiogenesis is inhibited. Such conditions include, but are not limitedto coronary artery disease, stroke, diabetes and chronic wounds.

Therapeutics for Orphan Indications and Diseases

The compounds and/or compositions of the present invention may be usedto treat orphan indications and/or diseases. Such diseases includeMarfan's syndrome. This syndrome is a connective tissue disorder,effecting bodily growth and development. Tissues and organs that aremost severely compromised include the heart, blood vessels, bones, eyes,lungs and connective tissue surrounding the spinal cord. Unfortunately,the effects can be life threatening. Marfan's syndrome is caused by agenetic mutation in the gene that produces fibrillin, a major componentof bodily connective tissue. Latent TGF-β binding protein (LTBP) is animportant regulator of TGF-β signaling that exhibits close identity tofibrillin protein family members. Functional LTBP is required forcontrolling the release of active TGF-β (Oklu, R. et al., The latenttransforming growth factor beta binding protein (LTBP) family. BiochemJ. 2000 Dec. 15; 352 Pt 3:601-10). In some embodiments, compounds and/orcompositions of the present invention are designed to alter the releaseprofile of TGF-β. In such embodiments, compounds and/or compositions maycomprise antibodies characterized as inhibitory antibodies.

In some embodiments, compounds and/or compositions of the presentinvention may be useful in the treatment of Camurati-Engelmann disease(CED). This disease primarily affects the bones, resulting in increasedbone density. Especially affected are the long bones of the legs andarms; however, the bones of the skill and hips can also be affected. Thedisease results in leg and arm pain as well as a variety of othersymptoms. CED is very rare, reported in approximately 200 individualsworldwide and is caused by a mutation in the TGF-β gene. TGF-β producedin the bodies of these individuals has a defective prodomain, leading tooveractive TGF-β signaling (Janssens, K. et al., Transforming growthfactor-beta 1 mutations in Camurati-Engelmann disease lead to increasedsignaling by altering either activation or secretion of the mutantprotein. J Biol Chem. 2003 Feb. 28; 278(9):7718-24. Epub 2002 Dec. 18).As described by Shi et al., (Shi, M. et al., Latent TGF-beta structureand activation. Nature. 2011 Jun. 15; 474(7351):343-9) among CEDmutations, Y81H disrupts an α2-helix residue that cradles the TGF-βfingers. The charge-reversal E169K and H222D mutations disrupt apH-regulated salt bridge between Glu 169 and His 222 in the dimerizationinterface of the prodomain. Residue Arg 218 is substantially buried: itforms a cation-π bond with Tyr 171 and salt bridges across the dimerinterface with residue Asp 226 of the ‘bowtie’ region of the growthfactor prodomain complex (GPC). Moreover, CED mutations in Cys 223 andCys 225 demonstrate the importance of disulphide bonds in the bowtieregion for holding TGF-β in inactive form. In this embodiment, compoundsand/or compositions of the present invention comprising one or moreinhibitory antibodies would serve to alleviate symptoms. In someembodiments, administration would be to the neonate subject.

Therapeutics for Immune and Autoimmune Diseases and Disorders

Compounds and/or compositions of the present invention may be used totreat immune and autoimmune disorders. Such disorders include, but arenot limited to Acute Disseminated Encephalomyelitis (ADEM), Acutenecrotizing hemorrhagic leukoencephalitis, Addison's disease,Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosingspondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome(APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmunedysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia,Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED),Autoimmune myocarditis, Autoimmune pancreatitis, Autoimmune retinopathy,Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease,Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease,Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease,Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronicinflammatory demyelinating polyneuropathy (CIDP), Chronic recurrentmultifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricialpemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome,Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis,CREST disease, Essential mixed cryoglobulinemia, Demyelinatingneuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease(neuromyelitis optica), Diabetes Type I, Discoid lupus, Dressler'ssyndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilicfasciitis, Erythema nodosum, Experimental allergic encephalomyelitis,Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Glomerulonephritis, Goodpasture's syndrome,Granulomatosis with Polyangiitis (GPA) see Wegener's, Graves' disease,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpesgestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura(ITP), IgA nephropathy, IgG4-related sclerosing disease,Immunoregulatory lipoproteins, Inclusion body myositis,Insulin-dependent diabetes (typel), Interstitial cystitis, Juvenilearthritis, Juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome,Large vessel vasculopathy, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopicpolyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer,Mucha-Habermann disease, Multiple endocrine neoplasia syndromes,Multiple sclerosis, Myositis, Myasthenia gravis, Narcolepsy,Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricialpemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (PediatricAutoimmune Neuropsychiatric Disorders Associated with Streptococcus),Paraneoplastic cerebellar degeneration, Paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turnersyndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheralneuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMSsyndrome, Polyarteritis nodosa, Type I, II, & III autoimmunepolyglandular syndromes, Polyendocrinopathies, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primarysclerosing cholangitis, Psoriasis, Psoriatic arthritis, IdiopathicPulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia,Raynauds phenomenon, Reactive arthritis, Reflex sympathetic dystrophy,Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome,Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis,Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren'ssyndrome, Small vessel vasculopathy, Sperm & testicular autoimmunity,Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac'ssyndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporalarteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome, Transverse myelitis, Tubular autoimmune disorder,Ulcerative colitis, Undifferentiated connective tissue disease (UCTD),Uveitis, Vesiculobullous dermatosis, Vasculitis, Vitiligo and Wegener'sgranulomatosis (also known as Granulomatosis with Polyangiitis (GPA)).

TGF-β plays an active role in leukocyte differentiation, proliferationand activation making it an important factor in immune and autoimmunediseases. Additionally, TGF-β promotes chemotaxis of leukocytes andinfluences adhesion molecule-mediated localization. A role for TGF-β incardiac, pulmonary and gastric inflammation has been demonstrated.Furthermore, SMAD3-deficient mice are prone to chronic mucosalinfections as a result of T-cell activation impairment and reducedmucosal immunity (Blobe, G. C. et al., Role of transforming growthfactor beta in human disease. N Engl J Med. 2000 May 4; 342(18):1350-8).As an immunosuppressant, TGF-β has been shown to both inhibit thefunction of inflammatory cells as well as enhance the function ofregulatory T cells. Recent studies have shown that the latent TGF-βgrowth factor prodomain complex (GPC) binds to regulatory T cellsthrough an interaction with the Glycoprotein-A repetitions anonymousprotein (GARP). In fact, GARP is necessary for TGF-β association with Tcells (Tran, D. Q. et al., GARP (LRRC32) is essential for the surfaceexpression of latent TGF-β on platelets and activated FOXP3⁺ regulatoryT cells. PNAS. 2009. 106(32):13445-50). This interaction provides theplatform necessary to release active TGF-β from the GPC in anintegrin-dependent manner (Wang, R. et al., GARP regulates thebioavailability and activation of TGF-β. Mol Biol Cell. 2012 March;23(6):1129-39. Epub 2012 Jan. 25). In some embodiments, compounds and/orcompositions of the present invention modulate the interaction betweenGARP and TGF-β. Such modulation may selectively modulate T cell activityfor treatment of disease (e.g. autoimmune disease and/or cancer.) Insome embodiments, compounds and/or compositions of the present inventionmay be used for the treatment of immune and/or autoimmune disorders. Insome embodiments, compounds and/or compositions of the present inventionmay specifically target GARP-bound GPC, GARP or the interaction sitebetween GARP and the GPC. In some embodiments, compounds and/orcompositions of the present invention comprising antibodies are designedto promote release of growth factors (including, but not limited toTGF-β) from GARP-bound GPCs while not affecting growth factor releasefrom LTBP-bound GPCs. Treatment of immune and autoimmune disorders withcompounds and/or compositions of the present invention may be incombination with standard of care (SOC) or synergistic combinations orwith companion diagnostics.

Therapeutics for Infectious Agents

In some embodiments, compounds and/or compositions of the presentinvention may be useful for treatment of infectious diseases and/ordisorders, for example, in subjects with one or more infections. In someembodiments, subjects have one or more infection or are at risk ofdeveloping one or more infection. As used herein, the term “infection”refers to a disease or condition in a host attributable to the presenceof one or more foreign organism or agent capable of reproduction withinthe host. Infections typically comprise breaching of one or more normalmucosal or other tissue barriers by one or more infectious organisms oragents. Subjects having one or more infection are subjects that compriseone or more objectively measurable infectious organisms or agentspresent in their body. Subjects at risk of having one or more infectionare subjects that are predisposed to developing one or more infection.Such subjects may include, for example, subjects with known or suspectedexposure to one or more infectious organisms or agents. In someembodiments, subjects at risk of having infections may also includesubjects with conditions associated with impaired abilities to mountimmune responses to infectious organisms and/or agents, e.g., subjectswith congenital and/or acquired immunodeficiency, subjects undergoingradiation therapy and/or chemotherapy, subjects with burn injuries,subjects with traumatic injuries and subjects undergoing surgery orother invasive medical or dental procedures.

Infections are broadly classified as bacterial, viral, fungal, and/orparasitic based on the category of infectious organisms and/or agentsinvolved. Other less common types of infection are also known in theart, including, e.g., infections involving rickettsiae, mycoplasmas, andagents causing scrapie, bovine spongiform encephalopathy (BSE), andprion diseases (e.g., kuru and Creutzfeldt-Jacob disease). Examples ofbacteria, viruses, fungi, and parasites which cause infection are wellknown in the art. An infection can be acute, subacute, chronic, orlatent, and it can be localized or systemic. As used herein, the term“chronic infection” refers to those infections that are not cleared bythe normal actions of the innate or adaptive immune responses andpersist in the subject for a long duration of time, on the order ofweeks, months, and years. A chronic infection may reflect latency of theinfectious agent, and may include periods in which no infectioussymptoms are present, i.e., asymptomatic periods. Examples of chronicinfections include, but are not limited to, HIV infection andherpesvirus infections. Furthermore, an infection can be predominantlyintracellular or extracellular during at least one phase of theinfectious organism's or agent's life cycle in the host.

Compounds and/or compositions of the present invention and additionaltherapeutic agents may be administered in combination in the samecomposition (e.g., parenterally), as part of a separate composition orby another method described herein.

Therapeutics for Eye Related Diseases, Disorders and/or Conditions

In some embodiments, compounds and/or compositions of the presentinvention may be useful in the treatment of diseases, disorders and/orconditions related to eyes. These may include, but are not limited toglaucoma, dry eye and/or corneal wound healing. In some embodiments,compounds and/or compositions may be useful in the treatment ofglaucoma. Evidence suggests that TGF-β2 is upregulated in glaucoma(Picht, G. et al., Transforming growth factor beta 2 levels in theaqueous humor in different types of glaucoma and the relation tofiltering bleb development. Graefes Arch Clin Exp Ophthalmol. 2001 March239(3):199-207; Tripathi, R. C. et al., Aqueous humor in glaucomatouseyes contains an increased level of TGF-β2. Exp Eye Res. 1994 December59(6):723-7.) This includes primary open-angle glaucoma and juvenileglaucoma. There is also evidence that TGF-β2 may induce senescence-likeeffects in human trabecular meshwork cells, which control intraocularpressure (often dysfunctional in glaucoma) (Yu, A. L. et al., TGF-β2induces senescence-associated changes in human trabecular meshworkcells. Invest Ophthalmol Vis Sci. 2010 November 51(11): 5718-23.) Insome embodiments, compounds and/or compositions of the present inventionmay be used to decrease the ratio of free TGF-β2 to GPC-bound (inactive)TGF-β2 in or around eye tissues affected by or related to glaucoma.TGF-β-related proteins may also impact on corneal wound healing (e.g.after surgical repair and/or LASIK treatment) (Huh, M. I. et al.,Distribution of TGF-β isoforms and signaling intermediates in cornealfibrotic wound repair. J Cell Biochem. 2009 Oct. 1. 108(2): 476-88;Sumioka, T. et al., Inhibitory effect of blocking TGF-beta/Smad signalon injury-induced fibrosis of corneal endothelium. Mol Vis. 2008;14:2272-81. Epub 2008 Dec. 11; Carrington, L. M. et al., Differentialregulation of key stages in early corneal wound healing by TGF-betaisoforms and their inhibitors. Invest Ophthalmol Vis Sci. 2006 May;47(5):1886-94.) Compounds and/or compositions of the present inventionmay be used to modulate TGF-β-related proteins in the cornea to enableand/or enhance wound healing. Such compounds and/or compositions wouldbe welcomed in the field where previous attempts have been unsuccessful.Mead et al (Mead, A. L. et al., Evaluation of anti-TGF-beta2 antibody asa new postoperative anti-scarring agent in glaucoma surgery. InvestOphthalmol Vis Sci. 2003 August; 44(8):3394-401) developed anti-TGF-β2antibodies to prevent scarring in eye tissues; however, results ofclinical trials were inconclusive. In some embodiments, compounds and/orcompositions of the present invention may be used to modulate TGF-β2levels (free versus GPC-bound) thereby providing an alternate method ofapproaching anti-scarring therapy.

Therapeutics for Cardiovascular Indications

In some embodiments, compounds and/or compositions of the presentinvention may be used to treat one or more cardiovascular indications,including, but not limited to cardiac hypertrophy. Cardiac hypertrophycomprises enlargement of the heart due, typically due to increased cellvolume of cardiac cells (Aurigemma 2006. N Engl J Med. 355(3):308-10.)Age-related cardiac hypertrophy may be due, in part, to reducedcirculating levels of GDF-11. A study by Loffredo et al (Loffredo etal., 2013. Cell. 153:828-39) found that fusion of the circulatory systembetween young and old mice had a protective effect with regard tocardiac hypertrophy. The study identified GDF-11 as a circulating factorthat decreased with age in mice and was able to show that itsadministration could also reduce cardiac hypertrophy. Some compoundsand/or compositions of the present invention may be used to treat and/orprevent cardiac atrophy. Such compounds and/or compositions may compriseGDF-11 agonists that elevate levels of circulating GDF-11, in some casesthrough enhancing the dissociation of GDF-11 growth factor from latentGPCs.

In some embodiments, compositions and methods of the invention may beused to treat one or more types of arterial disorders. Such disordersmay include, but are not limited to the development of aortic aneurysms.Aortic aneurysms may arise from a variety of causes, but most resultultimately in the overexpression of TGF-β2. A study by Boileau et al(Boileau et al., Nature Genetics Letters. 2012. 44(8):916-23, thecontents of which are herein incorporated by reference in theirentirety) uncovered causative mutations in TGF-β2 that were associatedwith some inherited forms of susceptibility to thoracic aortic disease.Interestingly, although the mutations were predicted to causehaploinsufficiency for TGF-β2, the aortic tissues of individuals withsuch mutations comprised increased levels of TGF-β2, as determined byimmunostaining. Similar findings were found in aortic tissues fromindividuals suffering from Marfans syndrome (Nataatmadja et al., 2006.)In some cases, compounds and/or compositions of the present inventionmay be used to reduce or prevent elevated TGF-β2 signaling in suchinstances thereby limiting aneurysm development and/or progression.

In some embodiments, animal models may be used to develop and testcompounds and/or compositions of the present invention for use in thetreatment of cardiovascular diseases, disorders and/or conditions. Insome cases, vascular injury models may be used. Such models may includeballoon injury models. In some cases, these may be carried out asdescribed in Smith et al., 1999. Circ Res. 84(10):1212-22, the contentsof which are herein incorporated by reference in their entirety.

Therapeutics Related to Muscle Disorders and/or Injuries

In some embodiments, compounds and/or compositions of the presentinvention may be used to treat one or more muscle disorders and/orinjuries. In some cases, such compounds and/or composition may include,but are not limited to antibodies that modulate GDF-8, GDF-11 and/oractivin activity. Muscle comprises about 40-50% of total body weight,making it the largest organ in the body. Muscle disorders may includecachexia (e.g. muscle wasting.) Muscle wasting may be associated with avariety of diseases and catabolic disorders (e.g. HIV/AIDS, cancer,cancer cachexia, renal failure, congestive heart failure, musculardystrophy, disuse atrophy, chronic obstructive pulmonary disease, motorneuron disease, trauma, neurodegenerative disease, infection, rheumatoidarthritis, immobilization, diabetes, etc.) In such disorders, GDF-8and/or activin signaling activity may contribute to muscle catabolism(Han et al., 2013. Int J Biochem Cell Biol. 45(10):2333-47; Lee., 2010.Immunol Endocr Metab Agents Med Chem. 10:183-94, the contents of each ofwhich are herein incorporated by reference in their entirety.) Othermuscle disorders may comprise sarcopenia. Sarcopenia is the progressiveloss of muscle and function associated with aging. In the elderly,sarcopenia can cause frailty, weakness, fatigue and loss of mobility(Morely. 2012. Family Practice. 29:i44-i48.) With the aged populationincreasing in numbers, sarcopenia is progressively becoming a moreserious public health concern. A study by Hamrick et al (Hamrick et al.,2010. 69(3):579-83) demonstrated that GDF-8 inhibition could repairmuscle in a mouse model of fibula osteotomoy comprising lateralcompartment muscle damage. Administration of GDF-8 propeptides wassufficient to increase muscle mass by nearly 20% as well as improvefracture healing. Some compounds and/or compositions of the presentinvention may be used to treat muscle diseases, disorders and/orinjuries by modulating GDF-8 activity. In some cases, compounds of thepresent invention may be GDF-8 signaling antagonists, preventing orreducing GDF-8 signaling activity.

Inclusion body myositis (IBM) is a disease characterized by progressivemuscle loss, typically occurring in mid- to late-life. The disease isthought to occur due to an autoimmune response to autoantigens in themuscle causing T-cell invasion of the muscle fiber and resulting inmyofiber destruction (Greenberg 2012. Curr Opin Neurol. 25(5):630-9.)Therapeutic compounds are being investigated, including Bimagrumab(BYM338; Novartis, Basel, Switzerland) an antibody that targets type IIactivin receptors, preventing GDF-8 and/or activin signal transduction,thereby stimulating muscle production and strengthening [see clinicaltrial number NCT01925209 entitled Efficacy and Safety ofBimagrumab/BYM338 at 52 Weeks on Physical Function, Muscle Strength,Mobility in sIBM Patients (RESILIENT)] Some compounds and/orcompositions of the present invention may be used to treat subjects withIBM. In some cases, such compounds and/or compositions may block GDF-8activity (e.g. through stabilization of GDF-8 GPCs.) In addition to IBM,BYM338 is being investigated for treatment of chronic obstructivepulmonary disease (COPD.) In some cases, compounds and/or compositionsof the present invention utilized for IBM treatment, may be used totreat COPD as well. In some cases, compounds and/or compositions of thepresent invention may be administered in combination and/or coordinationwith BYM338.

Therapeutics for Diabetes

Skeletal muscle uses and stores glucose for fuel. Due to this, skeletalmuscle is an important regulator of circulating glucose levels. Uptakeof glucose by muscle can be stimulated by either contraction or byinsulin stimulation (McPherron et al., 2013. Adipocyte. 2(2):92-8,herein incorporated by reference in its entirety). A recent study by Guoet al (Guo, et al., 2012. Diabetes 61(10):2414-23) found that when GDF-8receptor-deficient mice were crossed with A-ZIP/F1 mice (alipodistrophic mouse strain, used as a diabetic model) hybrid off-springshowed reduced levels of blood glucose and improved sensitivity toinsulin. Hyperphagia (excessive eating) was also reduced in these mice.In some embodiments, compound and/or compositions of the presentinvention may be used to treat diabetes and/or hyperphagia. Some suchtreatments may be used to reduce blood glucose and/or improve insulinsensitivity. In some cases, such treatments may comprise GDF-8 signalingantagosists, such as one or more antibodies that prevent dissociation ofGDF-8 from its prodomain.

Therapeutics for Gastro-Intestinal Diseases, Disorders and/or Conditions

In some embodiments, compositions and methods of the invention may beused to treat one or more types of gastro-intestinal (GI) disorders.Such disorders may include, but are not limited to inflammatory boweldisease (IBD) (e.g. Crohn's disease and ulcerative colitis.)

TGF-β2 may play a role in gut homeostasis and may have ananti-inflammatory role, protecting against GI-related disorders such asmucositis and certain forms of colitis. In one study, TGF-β2 was shownto suppress macrophage inflammatory responses in the developingintestine and protect against inflammatory mucosal injury (Maheshwari etal., 2011.) Interestingly, levels of TGF-β2 are high in breast milk,suggesting that TGF-β2 may function, in some cases, topically. Indeed,TGF-β2 in breast milk may attenuate inflammatory responses (Rautava etal., 2011.) Some compounds, compositions and/or methods of the presentinvention may be used to modulate GI TGF-β2 levels and/or activity inthe maintenance of homeostasis and/or in the management of GI-relateddisorders.

In some cases, models of GI-related diseases, disorders and/orconditions may be used to develop and/or test compounds and/orcompositions of the invention for treatment of GI-related diseases,disorders and/or conditions. In some cases, GI injury models may beused. Such injury models may include, but are not limited to2,4,6-trinitrobenzenesulfonic acid (TNBS) induced colitis models. Suchmodels may be carried out as described in Scheiffele, F. et al., 2002.Curr Protoc Immunol. Chapter 15:Unit 15.19, the contents of which areherein incorporated by reference in their entirety.

Veterinary Applications

In some embodiments, it is contemplated that compositions and methods ofthe invention will find utility in the area of veterinary care includingthe care and treatment of non-human vertebrates. As described herein,the term “vertebrate” includes all vertebrates including, but notlimited to fish, amphibians, birds, reptiles and mammals (including, butnot limited to alpaca, banteng, bison, camel, cat, cattle, deer, dog,donkey, gayal, goat, guinea pig, horse, llama, mice, monkeys, mule, pig,rabbit, rats, reindeer, sheep water buffalo, yak and humans.) As usedherein the term “non-human vertebrate” refers to any vertebrate with theexception of humans (i.e. Homo sapiens). Exemplary non-human vertebratesinclude wild and domesticated species such as companion animals andlivestock. Livestock include domesticated animals raised in anagricultural setting to produce materials such as food, labor, andderived products such as fiber and chemicals. Generally, livestockincludes all mammals, avians and fish having potential agriculturalsignificance. In particular, four-legged slaughter animals includesteers, heifers, cows, calves, bulls, cattle, swine and sheep.

Bioprocessing

In some embodiments, the present invention provides methods forproducing one or more biological products in host cells by contactingsuch cells with compounds and/or compositions of the present inventioncapable of modulating expression of target genes, or altering the levelof growth factor signaling molecules wherein such modulation oralteration enhances production of biological products. According to thepresent invention, bioprocessing methods may be improved by using one ormore compounds and/or compositions of the present invention. They mayalso be improved by supplementing, replacing or adding one or morecompounds and/or compositions.

Pharmaceutical Compositions

The pharmaceutical compositions described herein may be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

In some embodiments, pharmaceutical compositions comprise complexes ofcompounds and/or compositions of the present invention with GPCs. Insuch embodiments, complexes may be implanted at desired therapeuticsites where steady dissociation of growth factors from complexes mayoccur over a desired period of time. In some embodiments, implantationcomplexes may be carried out in association with sponge and/or bone-likematrices. Such implantations may include, but are not limited to dentalimplant sites and/or sites of bone repair.

In some embodiments, compounds and/or compositions of the presentinvention are made in furin-deficient cells. GPCs produced in such cellsmay be useful for treatment in areas where release is slowed due to thefact that furin cleavage in vivo is rate-limiting during GPC processing.In some embodiments, one or more tolloid and/or furin sites in GPCs aremutated, slowing the action of endogenous tolloid and/or furinproteases. In such embodiments, growth factor release may be slowed(e.g. at sites of implantation.)

Antibodies of the present invention, when formulated into compositionswith delivery/formulation agents or vehicles as described herein, mayexhibit increased bioavailability as compared to compositions lackingdelivery agents as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof a particular agent administered to a subject. Bioavailability may beassessed by measuring the area under the curve (AUC) or the maximumserum or plasma concentration (C_(max)) of the unchanged form of acompound following administration of the compound to a mammal. AUC is adetermination of the area under the curve plotting the serum or plasmaconcentration of a compound along the ordinate (Y-axis) against timealong the abscissa (X-axis). Generally, the AUC for a particularcompound may be calculated using methods known to those of ordinaryskill in the art and as described in G. S. Banker, Modern Pharmaceutics,Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York,Inc., 1996, the contents of which are herein incorporated by referencein their entirety.

C_(max) values are maximum concentrations of compounds achieved in serumor plasma of a subject following administration of compounds to thesubject. C_(max) values of particular compounds may be measured usingmethods known to those of ordinary skill in the art. As used herein, thephrases “increasing bioavailability” or “improving thepharmacokinetics,” refer to actions that may increase the systemicavailability of a compounds and/or compositions of the present invention(as measured by AUC, C_(max), or C_(min)) in a subject. In someembodiments, such actions may comprise co-administration with one ormore delivery agents as described herein. In some embodiments, thebioavailability of compounds and/or compositions may increase by atleast about 2%, at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95% or about 100%.

Therapeutic Window

Compounds and/or compositions of the present invention, when formulatedwith one or more delivery agents as described herein, may exhibitincreases in the therapeutic window of compound and/or compositionadministration as compared to the therapeutic window of compounds and/orcompositions administered without one or more delivery agents asdescribed herein. As used herein, the term “therapeutic window” refersto the range of plasma concentrations, or the range of levels oftherapeutically active substance at the site of action, with a highprobability of eliciting a therapeutic effect. In some embodiments,therapeutic windows of compounds and/or compositions whenco-administered with one or more delivery agent as described herein mayincrease by at least about 2%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95% or about 100%.

Volume of Distribution

Compounds and/or compositions of the present invention, when formulatedwith one or more delivery agents as described herein, may exhibit animproved volume of distribution (V_(dist)), e.g., reduced or targeted,relative to formulations lacking one or more delivery agents asdescribed herein. V_(dist) relates the amount of an agent in the body tothe concentration of the same agent in the blood or plasma. As usedherein, the term “volume of distribution” refers to the fluid volumethat would be required to contain the total amount of an agent in thebody at the same concentration as in the blood or plasma: V_(dist)equals the amount of an agent in the body/concentration of the agent inblood or plasma. For example, for a 10 mg dose of a given agent and aplasma concentration of 10 mg/L, the volume of distribution would be 1liter. The volume of distribution reflects the extent to which an agentis present in the extravascular tissue. Large volumes of distributionreflect the tendency of agents to bind to the tissue components ascompared with plasma proteins. In clinical settings, V_(dist) may beused to determine loading doses to achieve steady state concentrations.In some embodiments, volumes of distribution of compounds and/orcompositions of the present invention when co-administered with one ormore delivery agents as described herein may decrease at least about 2%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%.

Formulation, Administration, Delivery and Dosing

In some embodiments, compounds and/or compositions of the presentinvention are pharmaceutical compositions. As used herein, the term“pharmaceutical composition” refers to a compound and/or composition ofthe present invention that has been formulated with one or morepharmaceutically acceptable excipients. In some embodiments,pharmaceutical compositions may optionally comprise one or moreadditional active substances, e.g. therapeutically and/orprophylactically active substances. General considerations in theformulation and/or manufacture of pharmaceutical agents may be found,for example, in Remington: The Science and Practice of Pharmacy 21^(st)ed., Lippincott Williams & Wilkins, 2005 (incorporated herein byreference).

In some embodiments, compositions may be administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to compounds and/orcompositions of the present invention to be delivered as describedherein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to other subjects, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

In some embodiments, formulations of the pharmaceutical compositionsdescribed herein may be prepared by any method known or hereafterdeveloped in the art of pharmacology. In general, such preparatorymethods include the step of bringing active ingredients into associationwith excipients and/or one or more other accessory ingredients, andthen, if necessary and/or desirable, dividing, shaping and/or packagingproducts into desired single- or multi-dose units.

In some embodiments, pharmaceutical compositions of the presentinvention may be prepared, packaged, and/or sold in bulk, as single unitdoses, and/or as a plurality of single unit doses. As used herein, theterm “unit dose” refers to a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of active ingredient.Amounts of active ingredient are generally equal to the dosage of activeingredients which would be administered to subjects and/or convenientfractions of such a dosages such as, for example, one-half or one-thirdof such a dosages.

In some embodiments, relative amounts of active ingredients,pharmaceutically acceptable excipients, and/or any additionalingredients in pharmaceutical compositions of the present invention mayvary, depending upon identity, size, and/or condition of subjects to betreated and further depending upon routes by which compositions are tobe administered. By way of example, compositions may comprise betweenabout 0.1% and 100%, e.g., from about 0.5% to about 50%, from about 1%to about 30%, from about 5% to about 80% or at least 80% (w/w) activeingredient. In some embodiments, active ingredients are antibodiesdirected toward regulatory elements and/or GPCs.

Formulations

Compounds and/or compositions of the present invention may be formulatedusing one or more excipients to: (1) increase stability; (2) increasecell permeability; (3) permit the sustained or delayed release (e.g., ofcompounds and/or growth factors from such formulations); and/or (4)alter the biodistribution (e.g., target compounds to specific tissues orcell types). In addition to traditional excipients such as any and allsolvents, dispersion media, diluents, liquid vehicles, dispersion aids,suspension aids, surface active agents, isotonic agents, thickeningagents, emulsifying agents and preservatives, formulations of thepresent invention may comprise, without limitation, liposomes, lipidnanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides,proteins, cells transfected with the compounds and/or compositions ofthe present invention (e.g., for transplantation into subjects) andcombinations thereof.

Excipients

Various excipients for formulating pharmaceutical compositions andtechniques for preparing the composition are known in the art (seeRemington: The Science and Practice of Pharmacy, 21^(st) Edition, A. R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference).

In some embodiments, the use of conventional excipient media arecontemplated within the scope of the present disclosure, except insofaras any conventional excipient media may be incompatible with substancesand/or their derivatives, such as by producing any undesirablebiological effects or otherwise interacting in deleterious manners withany other component(s) of pharmaceutical compositions.

Formulations of pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include steps ofassociating active ingredients with excipients and/or other accessoryingredients.

Pharmaceutical compositions, in accordance with the present disclosure,may be prepared, packaged, and/or sold in bulk, as single unit doses,and/or as a plurality of single unit doses.

Relative amounts of active ingredients, pharmaceutically acceptableexcipients, and/or additional ingredients in pharmaceutical compositionsof the present disclosure may vary, depending upon identity, size,and/or condition of subjects being treated and further depending uponroutes by which pharmaceutical compositions may be administered.

In some embodiments, pharmaceutically acceptable excipient are at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%pure. In some embodiments, excipients are approved for use in humansand/or for veterinary use. In some embodiments, excipients are approvedby the United States Food and Drug Administration. In some embodiments,excipients are pharmaceutical grade. In some embodiments, excipientsmeet the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

In some embodiments, pharmaceutically acceptable excipients of thepresent invention may include, but are not limited to, inert diluents,dispersing and/or granulating agents, surface active agents and/oremulsifiers, disintegrating agents, binding agents, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Formulation Vehicles: Liposomes, Lipoplexes, and Lipid Nanoparticles

Compounds and/or compositions of the present invention may be formulatedusing one or more liposomes, lipoplexes and/or lipid nanoparticles. Insome embodiments, pharmaceutical compositions comprise liposomes.Liposomes are artificially-prepared vesicles which may primarily becomposed of a lipid bilayer and may be used as delivery vehicles for theadministration of nutrients and pharmaceutical formulations. Liposomesmay be of different sizes such as, but not limited to, multilamellarvesicles (MLVs) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, small unicellular vesicle (SUVs) which may be smaller than50 nm in diameter and large unilamellar vesicle (LUVs) which may bebetween 50 and 500 nm in diameter. Liposome components may include, butare not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may comprise low or highpH. In some embodiments, liposome pH may be varied in order to improvedelivery of pharmaceutical formulations.

In some embodiments, liposome formation may depend on physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped, liposomal ingredients, the nature of the mediumin which lipid vesicles are dispersed, the effective concentration ofentrapped substances, potential toxicity of entrapped substances,additional processes involved during the application and/or delivery ofvesicles, optimization size, polydispersity, shelf-life of vesicles forthe intended application, batch-to-batch reproducibility and possibilityof large-scale production of safe and efficient liposomal products.

In some embodiments, formulations may be assembled or compositionsaltered such that they are passively or actively directed to differentcell types in vivo.

In some embodiments, formulations may be selectively targeted throughexpression of different ligands on formulation surfaces as exemplifiedby, but not limited by, folate, transferrin, N-acetylgalactosamine(GalNAc), and antibody targeted approaches.

In some embodiments, pharmaceutical compositions of the presentinvention may be formulated with liposomes, lipoplexes and/or lipidnanoparticles to improve efficacy of function. Such formulations may beable to increase cell transfection by pharmaceutical compositions. Insome embodiments, liposomes, lipoplexes, or lipid nanoparticles may beused to increase pharmaceutical composition stability.

In some embodiments, liposomes are specifically formulated forpharmaceutical compositions comprising one or more antibodies. Suchliposomes may be prepared according to techniques known in the art, suchas those described by Eppstein et al. (Eppstein, D. A. et al.,Biological activity of liposome-encapsulated murine interferon gamma ismediated by a cell membrane receptor. Proc Natl Acad Sci USA. 1985 June;82(11):3688-92); Hwang et al. (Hwang, K. J. et al., Hepatic uptake anddegradation of unilamellar sphingomyelin/cholesterol liposomes: akinetic study. Proc Natl Acad Sci USA. 1980 July; 77(7):4030-4); U.S.Pat. No. 4,485,045 and U.S. Pat. No. 4,544,545. Production of liposomeswith sustained circulation time are also described in U.S. Pat. No.5,013,556.

In some embodiments, liposomes of the present invention comprisingantibodies may be generated using reverse phase evaporation utilizinglipids such as phosphatidylcholine, cholesterol as well asphosphatidylethanolamine that have been polyethylene glycol-derivatized.Filters with defined pore size are used to extrude liposomes of thedesired diameter. In another embodiment, compounds and/or compositionsof the present invention may be conjugated to external surfaces ofliposomes by disulfide interchange reactions as is described by Martinet al. (Martin, F. J. et al., Irreversible coupling of immunoglobulinfragments to preformed vesicles. An improved method for liposometargeting. J Biol Chem. 1982 Jan. 10; 257(1):286-8).

Formulation Vehicles: Polymers and Nanoparticles

Compounds and/or compositions of the present invention may be formulatedusing natural and/or synthetic polymers. Non-limiting examples ofpolymers which may be used for delivery include, but are not limited toDMRI/DOPE, poloxamer, chitosan, cyclodextrin, andpoly(lactic-co-glycolic acid) (PLGA) polymers. In some embodiments,polymers may be biodegradable.

In some embodiments, polymer formulation may permit sustained and/ordelayed release of compounds and/or compositions (e.g., followingintramuscular and/or subcutaneous injection). Altered release profilefor compounds and/or compositions of the present invention may resultin, for example, compound release over an extended period of time.Polymer formulations may also be used to increase the stability ofcompounds and/or compositions of the present invention.

In some embodiments, polymer formulations may be selectively targetedthrough expression of different ligands as exemplified by, but notlimited by, folate, transferrin, and N-acetylgalactosamine (GalNAc)(Benoit, D. S. et al., Synthesis of folate-functionalized RAFT polymersfor targeted siRNA delivery. Biomacromolecules. 2011 12:2708-14; Rozema,D. B. et al., Dynamic polyconjugates for targeted in vivo delivery ofsiRNA to hepatocytes. Proc Natl Acad Sci USA. 2007 104:12982-12887;Davis, M. E. et al., The first targeted delivery of siRNA in humans viaa self-assembling, cyclodextrin polymer-based nanoparticle: from conceptto clinic. Mol Pharm. 2009 6:659-668; Davis, M. E. et al., Evidence ofRNAi in humans from systemically administered siRNA via targetednanoparticles. Nature. 2010. 464:1067-70; the contents of each of whichare herein incorporated by reference in their entirety.)

Compounds and/or compositions of the present invention may be formulatedas nanoparticles using combinations of polymers, lipids, and/or otherbiodegradable agents, such as, but not limited to, calcium phosphates.In some embodiments, components may be combined in core-shells, hybrids,and/or layer-by-layer architectures, to allow for fine-tuning ofnanoparticle structure, so delivery may be enhanced. For antibodies ofthe present invention, systems based on poly(2-(methacryloyloxy)ethylphosphorylcholine)-block-(2-(diisopropylamino)ethyl methacrylate),(PMPC-PDPA), a pH sensitive diblock copolymer that self-assembles toform nanometer-sized vesicles, also known as polymersomes, atphysiological pH may be used. These polymersomes have been shown tosuccessfully deliver relatively high antibody payloads within livecells. (Massignani, M. et al., Cellular delivery of antibodies:effective targeted subcellular imaging and new therapeutic tool. NatureProceedings. 2010. p 1-17.)

In some embodiments, PEG-charge-conversional polymers (Pitella, F. etal., Enhanced endosomal escape of siRNA-incorporating hybridnanoparticles from calcium phosphate and PEG-block charge-conversionalpolymer for efficient gene knockdown with negligible cytotoxicity.Biomaterials. 2011 32:3106-14) may be used to form nanoparticles fordelivery of compounds and/or compositions of the present invention. Insome embodiments, PEG-charge-conversional polymers may improve uponPEG-polyanion block copolymers by being cleaved into polycations atacidic pH, thus enhancing endosomal escape.

In some embodiments, complexation, delivery and/or internalization ofpolymeric nanoparticles may be precisely controlled by altering chemicalcompositions in both core and shell nanoparticle components (Siegwart,D. J. et al., Combinatorial synthesis of chemically diverse core-shellnanoparticles for intracellular delivery. Proc Natl Acad Sci USA. 2011108:12996-3001).

In some embodiments, matrices of poly(ethylene-co-vinyl acetate), areused to deliver compounds and/or compositions of the invention. Suchmatrices have bee described by others (Sherwood, J. K. et al.,Controlled antibody delivery systems. Nature Biotechnology. 1992.10:1446-9.)

Antibody Formulations

Antibodies of the present invention may be formulated for intravenousadministration or extravascular administration (Daugherty, et al.,Formulation and delivery issues for monoclonal antibody therapeutics.Adv Drug Deliv Rev. 2006 Aug. 7; 58(5-6):686-706 and US patentapplication publication number US2011/0135570, the contents of each ofwhich are herein incorporated by reference in their entirety).Extravascular administration routes may include, but are not limited tosubcutaneous administration, intraperitoneal administration,intracerebral administration, intraocular administration, intralesionaladministration, topical administration and intramuscular administration.

In some embodiments, antibody structures may be modified to improveeffectiveness as therapeutics. Improvements may include, but are notlimited to improved thermodynamic stability, reduced Fc receptor bindingproperties and/or imporved folding efficiency. Modifications mayinclude, but are not limited to amino acid substitutions, glycosylation,palmitoylation and/or protein conjugation.

In some embodiments, antibodies of the present invention may beformulated with antioxidants to reduce antibody oxidation. Antibodies ofthe present invention may also be formulated with additives to reduceprotein aggregation. Such additives may include, but are not limited toalbumin, amino acids, sugars, urea, guanidinium chloride, polyalchohols,polymers (such as polyethylene glycol and dextrans), surfactants(including, but not limited to polysorbate 20 and polysorbate 80) oreven other antibodies.

In some embodiments, antibodies of the present invention may beformulated to reduce the impact of water on antibody structure andfunction. Antibody preparartions in such formulations may be may belyophilized. Formulations subject to lyophilization may includecarbohydrates or polyol compounds to protect and/or stabilize antibodystructure. Such compounds may include, but are not limited to sucrose,trehalose and mannitol.

In some embodiments, antibodies of the present invention may beformulated with polymers. In some embodiments, polymer formulations maycomprise hydrophobic polymers. Such polymers may be microspheresformulated with polylactide-co-glycolide through solid-in-oil-in-waterencapsulation methods. In some embodiments, microspheres comprisingethylene-vinyl acetate copolymer may also be used for antibody deliveryand/or to extend the time course of antibody release at sites ofdelivery. In some embodiments, polymers may be aqueous gels. Such gelsmay, for example, comprise carboxymethylcellulose. In some embodiments,aqueous gels may also comprise hyaluronic acid hydrogels. In someembodiments, antibodies may be covalently linked to such gels throughhydrazone linkages that allow for sustained delivery in tissues,including but not limited to tissues of the central nervous system.

Formulation Vehicles: Peptides and Proteins

Compounds and/or compositions of the present invention may be formulatedwith peptides and/or proteins. In some embodiments, peptides such as,but not limited to, cell penetrating peptides and/or proteins/peptidesthat enable intracellular delivery may be used to deliver pharmaceuticalformulations. Non-limiting examples of a cell penetrating peptides whichmay be used with pharmaceutical formulations of the present inventioninclude cell-penetrating peptide sequences attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g. Caron, N.J. et al., Intracellular delivery of aTat-eGFP fusion protein into muscle cells. Mol Ther. 2001. 3(3):310-8;Langel, U., Cell-Penetrating Peptides: Processes and Applications, CRCPress, Boca Raton Fla., 2002; El-Andaloussi, S. et al., Cell-penetratingpeptides: mechanisms and applications. Curr Pharm Des. 2003.11(28):3597-611; and Deshayes, S. et al., Cell-penetrating peptides:tools for intracellular delivery of therapeutics. Cell Mol Life Sci.2005. 62(16):1839-49, the contents of each of which are hereinincorporated by reference in their entirety.) Compounds and/orcompositions of the present invention may also be formulated to includecell penetrating agents, e.g., liposomes, which enhance delivery of thecompositions to intracellular spaces. Compounds and/or compositions ofthe present invention may be complexed with peptides and/or proteinssuch as, but not limited to, peptides and/or proteins from AileronTherapeutics (Cambridge, Mass.) and Permeon Biologics (Cambridge, Mass.)in order to enable intracellular delivery (Cronican, J. J. et al.,Potent delivery of functional proteins into mammalian cells in vitro andin vivo using a supercharged protein. ACS Chem Biol. 2010. 5:747-52;McNaughton, B. R. et al., Mammalian cell penetration, siRNAtransfection, and DNA transfection by supercharged proteins. Proc NatlAcad Sci, USA. 2009. 106:6111-6; Verdine, G. L. et al., Stapled peptidesfor intracellular drug targets. Methods Enzymol. 2012. 503:3-33; thecontents of each of which are herein incorporated by reference in theirentirety).

In some embodiments, the cell-penetrating polypeptides may comprisefirst and second domains. First domains may comprise superchargedpolypeptides. Second domains may comprise protein-binding partner. Asused herein, protein-binding partners may include, but are not limitedto, antibodies and functional fragments thereof, scaffold proteinsand/or peptides. Cell-penetrating polypeptides may further compriseintracellular binding partners for protein-binding partners. In someembodiments, cell-penetrating polypeptides may be capable of beingsecreted from cells where compounds and/or compositions of the presentinvention may be introduced.

Compositions of the present invention comprising peptides and/orproteins may be used to increase cell transfection and/or altercompound/composition biodistribution (e.g., by targeting specifictissues or cell types).

Formulation Vehicles: Cells

Cell-based formulations of compounds and/or compositions of the presentinvention may be used to ensure cell transfection (e.g., in cellularcarriers) or to alter biodistribution (e.g., by targeting cell carriersto specific tissues or cell types.)

Cell Transfer Methods

A variety of methods are known in the art and suitable for introductionof nucleic acids or proteins into cells, including viral and non-viralmediated techniques. Examples of typical non-viral mediated techniquesinclude, but are not limited to, electroporation, calcium phosphatemediated transfer, nucleofection, sonoporation, heat shock,magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediatedtransfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) andthe like) or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofcell plasma membranes. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon, C. S. et al.,Ultrasound-mediated gene delivery. Expert Opin Drug Deliv. 20107:321-30; Postema, M. et al., Ultrasound-directed drug delivery. CurrPharm Biotechnol. 2007 8:355-61; Newman, C. M. et al., Gene therapyprogress and prospects: ultrasound for gene transfer. Gene Ther. 2007.14(6):465-75; the contents of each of which are herein incorporated byreference in their entirety). Sonoporation methods are known in the artand are also taught for example as they relate to bacteria in US Patentapplication publication US2010/0196983 and as it relates to other celltypes in, for example, US Patent application publication US2010/0009424,the contents of each of which are incorporated herein by reference intheir entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre, F. M. et al.,Nucleic acids electrotransfer in vivo: mechanisms and practical aspects.Curr Gene Ther. 2010 10:267-80; Chiarella, P. et al., Application ofelectroporation in DNA vaccination protocols. Curr Gene Ther. 2010.10:281-6; Hojman, P., Basic principles and clinical advancements ofmuscle electrotransfer. Curr Gene Ther. 2010 10:128-38; the contents ofeach of which are herein incorporated by reference in their entirety).In some embodiments, compounds and/or compositions of the presentinvention may be delivered by electroporation.

Administration and Delivery

Compounds and/or compositions of the present invention may beadministered by any of the standard methods or routes known in the art.Such methods may include any route which results in a therapeuticallyeffective outcome. These include, but are not limited to enteral,gastroenteral, epidural, oral, transdermal, epidural (peridural),intracerebral (into the cerebrum), intracerebroventricular (into thecerebral ventricles), epicutaneous (application onto the skin),intradermal, (into the skin itself), subcutaneous (under the skin),nasal administration (through the nose), intravenous (into a vein),intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compounds and/or compositions of the present invention may beadministered in ways which allow them to cross the blood-brain barrier,vascular barriers, or other epithelial barriers. Methods of formulationand administration may include any of those disclosed in US Pub. No.2013/0122007, U.S. Pat. No. 8,415,459 or International Pub. No. WO2011/151432, the contents of each of which are herein incorporated byreference in their entirety. Non-limiting routes of administration forcompounds and/or compositions of the present invention are describedbelow.

Parenteral and Injectable Administration

In some embodiments, compounds and/or compositions of the presentinvention may be administered parenterally. Liquid dosage forms for oraland parenteral administration include, but are not limited to,pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and/or elixirs. In addition to active ingredients,liquid dosage forms may comprise inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and/or perfuming agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMOPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof. In other embodiments, surfactants are includedsuch as hydroxypropylcellulose.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations may be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of active ingredients, it is oftendesirable to slow the absorption of active ingredients from subcutaneousor intramuscular injections. This may be accomplished by the use ofliquid suspensions of crystalline or amorphous material with poor watersolubility. The rate of absorption of active ingredients depends uponthe rate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

In some embodiments, compounds and/or compositions of the presentinvention may be administered rectally and/or vaginally. Compositionsfor rectal or vaginal administration are typically suppositories whichcan be prepared by mixing compositions with suitable non-irritatingexcipients such as cocoa butter, polyethylene glycol or a suppositorywax which are solid at ambient temperature but liquid at bodytemperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

In some embodiments, compounds and/or compositions of the presentinvention may be administered orally. Solid dosage forms for oraladministration include capsules, tablets, pills, powders, and granules.In such solid dosage forms, an active ingredient is mixed with at leastone inert, pharmaceutically acceptable excipient such as sodium citrateor dicalcium phosphate and/or fillers or extenders (e.g. starches,lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g.carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents(e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate), solution retarding agents(e.g. paraffin), absorption accelerators (e.g. quaternary ammoniumcompounds), wetting agents (e.g. cetyl alcohol and glycerolmonostearate), absorbents (e.g. kaolin and bentonite clay), andlubricants (e.g. talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), and mixtures thereof. Inthe case of capsules, tablets and pills, the dosage form may comprisebuffering agents.

Topical or Transdermal Administration

As described herein, compounds and/or compositions of the presentinvention may be formulated for administration topically. The skin maybe an ideal target site for delivery as it is readily accessible. Threeroutes are commonly considered to deliver compounds and/or compositionsof the present invention to the skin: (i) topical application (e.g. forlocal/regional treatment and/or cosmetic applications); (ii) intradermalinjection (e.g. for local/regional treatment and/or cosmeticapplications); and (iii) systemic delivery (e.g. for treatment ofdermatologic diseases that affect both cutaneous and extracutaneousregions). Compounds and/or compositions of the present invention can bedelivered to the skin by several different approaches known in the art.

In some embodiments, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of compounds and/or compositions of the present inventiondescribed herein to allow users to perform multiple treatments.

Dosage forms for topical and/or transdermal administration may includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants and/or patches. Generally, active ingredients are admixedunder sterile conditions with pharmaceutically acceptable excipientsand/or any needed preservatives and/or buffers. Additionally, thepresent invention contemplates the use of transdermal patches, whichoften have the added advantage of providing controlled delivery ofcompounds and/or compositions of the present invention to the body. Suchdosage forms may be prepared, for example, by dissolving and/ordispensing compounds and/or compositions in the proper medium.Alternatively or additionally, rates may be controlled by eitherproviding rate controlling membranes and/or by dispersing compoundsand/or compositions in a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, compounds and/or compositionsof the present invention are formulated in depots for extended release.Generally, specific organs or tissues (“target tissues”) are targetedfor administration.

In some aspects of the invention, compounds and/or compositions of thepresent invention are spatially retained within or proximal to targettissues. Provided are method of providing compounds and/or compositionsto target tissues of mammalian subjects by contacting target tissues(which comprise one or more target cells) with compounds and/orcompositions under conditions such that they are substantially retainedin target tissues, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80,85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissues. Advantageously, retentionis determined by measuring the amount of compounds and/or compositionsthat enter one or more target cells. For example, at least 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.9%, 99.99% or greater than 99.99% of compounds and/or compositionsadministered to subjects are present intracellularly at a period of timefollowing administration. For example, intramuscular injection tomammalian subjects may be performed using aqueous compositionscomprising compounds and/or compositions of the present invention andone or more transfection reagent, and retention is determined bymeasuring the amount of compounds and/or compositions present in musclecells.

Certain aspects of the invention are directed to methods of providingcompounds and/or compositions of the present invention to a targettissues of mammalian subjects, by contacting target tissues (comprisingone or more target cells) with compounds and/or compositions underconditions such that they are substantially retained in such targettissues. Compounds and/or compositions comprise enough active ingredientsuch that the effect of interest is produced in at least one targetcell. In some embodiments, compounds and/or compositions generallycomprise one or more cell penetration agents, although “naked”formulations (such as without cell penetration agents or other agents)are also contemplated, with or without pharmaceutically acceptablecarriers.

In some embodiments, the amount of a growth factor present in cells in atissue is desirably increased. Preferably, this increase in growthfactor is spatially restricted to cells within the target tissue. Thus,provided are methods of increasing the amount of growth factor ofinterest in tissues of mammalian subjects. In some embodiments,formulations are provided comprising compounds and/or compositionscharacterized in that the unit quantity provided has been determined toproduce a desired level of growth factor of interest in a substantialpercentage of cells contained within predetermined volumes of targettissue.

In some embodiments, formulations comprise a plurality of differentcompounds and/or compositions, where one or more than one targetsbiomolecules of interest. Optionally, formulations may also comprisecell penetration agents to assist in the intracellular delivery ofcompounds and/or compositions. In such embodiments, determinations aremade of compound and/or composition dose required to target biomoleculesof interest in substantial percentages of cells contained withinpredetermined volumes of the target tissue (generally, without targetingbiomolecules of interest in adjacent or distal tissues.) Determineddoses are then introduced directly into subject tissues. In someembodiments, the invention provides for compounds and/or compositions tobe delivered in more than one administration or by split doseadministration.

Pulmonary Administration

In some embodiments, compounds and/or compositions of the presentinvention may be prepared, packaged, and/or sold in formulationssuitable for pulmonary administration. In some embodiments, suchadministration is via the buccal cavity. In some embodiments,formulations may comprise dry particles comprising active ingredients.In such embodiments, dry particles may have a diameter in the range fromabout 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. In someembodiments, formulations may be in the form of dry powders foradministration using devices comprising dry powder reservoirs to whichstreams of propellant may be directed to disperse such powder. In someembodiments, self propelling solvent/powder dispensing containers may beused. In such embodiments, active ingredients may be dissolved and/orsuspended in low-boiling propellant in sealed containers. Such powdersmay comprise particles wherein at least 98% of the particles by weighthave diameters greater than 0.5 nm and at least 95% of the particles bynumber have diameters less than 7 nm. Alternatively, at least 95% of theparticles by weight have a diameter greater than 1 nm and at least 90%of the particles by number have a diameter less than 6 nm. Dry powdercompositions may include a solid fine powder diluent such as sugar andare conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generallypropellants may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition.Propellants may further comprise additional ingredients such as liquidnon-ionic and/or solid anionic surfactant and/or solid diluent (whichmay have particle sizes of the same order as particles comprising activeingredients).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide active ingredients in the form of droplets of solution and/orsuspension. Such formulations may be prepared, packaged, and/or sold asaqueous and/or dilute alcoholic solutions and/or suspensions, optionallysterile, comprising active ingredients, and may conveniently beadministered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

In some embodiments, compounds and/or compositions of the presentinvention may be administered nasaly and/or intranasaly. In someembodiments, formulations described herein as being useful for pulmonarydelivery may also be useful for intranasal delivery. In someembodiments, formulations for intranasal administration comprise acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such formulations are administeredin the manner in which snuff is taken, i.e. by rapid inhalation throughthe nasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise powdersand/or an aerosolized and/or atomized solutions and/or suspensionscomprising active ingredients. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may comprise average particleand/or droplet sizes in the range of from about 0.1 nm to about 200 nm,and may further comprise one or more of any additional ingredientsdescribed herein.

Ophthalmic or Otic Administration

In some embodiments, compounds and/or compositions of the presentinvention may be prepared, packaged, and/or sold in formulationssuitable for ophthalmic and/or otic administration. Such formulationsmay, for example, be in the form of eye and/or ear drops including, forexample, a 0.1/1.0% (w/w) solution and/or suspension of the activeingredient in aqueous and/or oily liquid excipients. Such drops mayfurther comprise buffering agents, salts, and/or one or more other ofany additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise active ingredients in microcrystalline form and/or inliposomal preparations. Subretinal inserts may also be used as forms ofadministration.

Payload Administration: Detectable Agents and Therapeutic Agents

In some embodiments, compounds and/or compositions of the presentinvention may be used in a number of different scenarios in whichdelivery of a substance (the “payload”) to a biological target isdesired, for example delivery of detectable substances for detection ofthe target, or delivery of therapeutic and/or diagnostic agents.Detection methods may include, but are not limited to, both in vitro andin vivo imaging methods, e.g., immunohistochemistry, bioluminescenceimaging (BLI), Magnetic Resonance Imaging (MM), positron emissiontomography (PET), electron microscopy, X-ray computed tomography, Ramanimaging, optical coherence tomography, absorption imaging, thermalimaging, fluorescence reflectance imaging, fluorescence microscopy,fluorescence molecular tomographic imaging, nuclear magnetic resonanceimaging, X-ray imaging, ultrasound imaging, photoacoustic imaging, labassays, or in any situation where tagging/staining/imaging is required.

In some embodiments, compounds and/or compositions may be designed toinclude both linkers and payloads in any useful orientation. Forexample, linkers having two ends may be used to attach one end to thepayload and the other end to compounds and/or compositions. Compoundsand/or compositions of the present invention may include more than onepayload. In some embodiments, compounds and/or compositions may compriseone or more cleavable linker. In some embodiments, payloads may beattached to compounds and/or compositions via a linker and may befluorescently labeled for in vivo tracking, e.g. intracellularly.

In some embodiments, compounds and/or compositions of the presentinvention may be used in reversible drug delivery into cells.

Compounds and/or compositions of the present invention may be used inintracellular targeting of payloads, e.g., detectable or therapeuticagents, to specific organelles. In addition, compounds and/orcompositions of the present invention may be used to deliver therapeuticagents to cells or tissues, e.g., in living animals. For example, thecompounds and/or compositions described herein may be used to deliverchemotherapeutic agents to kill cancer cells. Compounds and/orcompositions may be attached to therapeutic agents through one or morelinkers may facilitate membrane permeation allowing therapeutic agentsto travel into cells to reach intracellular targets.

In some embodiments, payloads may be a therapeutic agent such as acytotoxins, radioactive ions, chemotherapeutics, or other therapeuticagents. Cytotoxins and/or cytotoxic agents may include any agents thatmay be detrimental to cells. Examples include, but are not limited to,taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, the contents of each of which are incorporatedherein by reference in their entirety), and analogs or homologs thereof.Radioactive ions include, but are not limited to iodine (e.g., ¹²⁵iodineor ¹³¹iodine), ⁸⁹strontium, phosphorous, palladium, cesium, iridium,phosphate, cobalt, ⁹⁰yttrium, ¹⁵³samarium, and praseodymium. Othertherapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, payloads may be detectable agents, such as variousorganic small molecules, inorganic compounds, nanoparticles, enzymes orenzyme substrates, fluorescent materials, luminescent materials (e.g.,luminol), bioluminescent materials (e.g., luciferase, luciferin, andaequorin), chemiluminescent materials, radioactive materials (e.g., ¹⁸F,⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl, ¹²⁵I, ³⁵S, ¹⁴C, ³H, or^(99m)Tc (e.g., as pertechnetate (technetate(VII), TcO₄ ⁻)), andcontrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium(e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide(SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmallsuperparamagnetic iron oxide (USPIO)), manganese chelates (e.g.,Mn-DPDP), barium sulfate, iodinated contrast media (iohexol),microbubbles, or perfluorocarbons). Such optically-detectable labelsinclude for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red), N,N,N′,Nletramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, andtetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectableprecursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (MA), and Western blot analysis.

Combinations

In some embodiments, compounds and/or compositions of the presentinvention may be used in combination with one or more other therapeutic,prophylactic, diagnostic, or imaging agents. By “in combination with,”it is not intended to imply that the agents must be administered at thesame time and/or formulated for delivery together, although thesemethods of delivery are within the scope of the present disclosure.Compounds and/or compositions of the present invention may beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. In general, each agentwill be administered at a dose and/or on a time schedule determined forthat agent. In some embodiments, the present disclosure encompasses thedelivery of pharmaceutical, prophylactic, diagnostic, or imagingcompositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body.

In some cases, compounds and/or compositions of the present inventionmay be combined with one or more therapeutic agents known in the art.Such agents may include BYM338 (Novartis, Basel, Switzerland) whereinadministration may comprise any of the methods disclosed in clinicaltrial number NCT01925209 entitled Efficacy and Safety ofBimagrumab/BYM338 at 52 Weeks on Physical Function, Muscle Strength,Mobility in sIBM Patients (RESILIENT). Other agents that may be used incombination with compounds and/or compositions of the present inventionmay include any of those disclosed in US Pub. No. 2013/0122007, U.S.Pat. No. 8,415,459 or International Pub. No. WO 2011/151432, thecontents of each of which are herein incorporated by reference in theirentirety.

Dosing and Dosage Forms

The present disclosure encompasses delivery of compounds and/orcompositions of the present invention for any of therapeutic,pharmaceutical, diagnostic or imaging by any appropriate route takinginto consideration likely advances in the sciences of drug delivery.Delivery may be naked or formulated.

Naked Delivery

Compounds and/or compositions of the present invention may be deliveredto cells, tissues, organs and/or organisms in naked form. As used hereinin, the term “naked” refers to compounds and/or compositions deliveredfree from agents or modifications which promote transfection orpermeability. The naked compounds and/or compositions may be deliveredto the cells, tissues, organs and/or organisms using routes ofadministration known in the art and described herein. In someembodiments, naked delivery may include formulation in a simple buffersuch as saline or PBS.

Formulated Delivery

In some embodiments, compounds and/or compositions of the presentinvention may be formulated, using methods described herein.Formulations may comprise compounds and/or compositions which may bemodified and/or unmodified. Formulations may further include, but arenot limited to, cell penetration agents, pharmaceutically acceptablecarriers, delivery agents, bioerodible or biocompatible polymers,solvents, and/or sustained-release delivery depots. Formulations of thepresent invention may be delivered to cells using routes ofadministration known in the art and described herein.

Compositions may also be formulated for direct delivery to organs ortissues in any of several ways in the art including, but not limited to,direct soaking or bathing, via a catheter, by gels, powder, ointments,creams, gels, lotions, and/or drops, by using substrates such as fabricor biodegradable materials coated or impregnated with compositions, andthe like.

Dosing

The present invention provides methods comprising administering one ormore compounds and/or compositions to subjects in need thereof.Compounds and/or compositions of the present invention, or prophylacticcompositions thereof, may be administered to subjects using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging diseases, disorders and/or conditions. The exactamount required will vary from subject to subject, depending on species,age and/or general subject condition, severity of disease, particularcomposition, mode of administration, mode of activity, and the like.Compositions in accordance with the invention are typically formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage ofcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or appropriateimaging dose level for any particular patient will depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, fromabout 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic, diagnostic, prophylactic, or imagingeffect. The desired dosage may be delivered three times a day, two timesa day, once a day, every other day, every third day, every week, everytwo weeks, every three weeks, or every four weeks. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

According to the present invention, compounds and/or compositions of thepresent invention may be administered in split-dose regimens. As usedherein, a “split dose” is the division of single unit dose or totaldaily dose into two or more doses, e.g., two or more administrations ofthe single unit dose. As used herein, a “single unit dose” is a dose ofany therapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event. As used herein, a“total daily dose” is an amount given or prescribed in a 24 hour period.In some embodiments, compounds and/or compositions of the presentinvention may be administered as a single unit dose. In someembodiments, compounds and/or compositions of the present invention maybe administered to subjects in split doses. In some embodiments,compounds and/or compositions of the present invention may be formulatedin buffer only or in formulations described herein. Pharmaceuticalcompositions described herein may be formulated into dosage formsdescribed herein, such as a topical, intranasal, intratracheal, orinjectable (e.g., intravenous, intraocular, intravitreal, intramuscular,intracardiac, intraperitoneal, subcutaneous). General considerations inthe formulation and/or manufacture of pharmaceutical agents may befound, for example, in Remington: The Science and Practice of Pharmacy21^(st) ed., Lippincott Williams & Wilkins, 2005 (incorporated herein byreference).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose and/or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Assays

In some embodiments, recombinant proteins (including, but not limited tochimeric proteins) disclosed herein and/or antibodies directed to suchproteins may be developed using assays described herein. In someembodiments, recombinant proteins (including, but not limited tochimeric proteins) disclosed herein and/or antibodies directed to suchproteins may be used in assays to develop other recombinant proteinsand/or antibodies of the present invention.

Binding Assays

In some embodiments, the present invention provides binding assays. Asused herein, the term “binding assay” refers to an assay used to assessthe ability of two or more factors to associate. Such assays may assessthe ability of a desired antigen to bind a desired antibody and then useone or more detection methods to detect binding. Binding assays of theinvention may include, but are not limited to surface Plasmonresonance-based assays, ELISAs and FACS-based assays. Binding assays ofthe invention may comprise the use of one or more recombinant proteinsdescribed herein, including, but not limited to any TGF-β family memberproteins, any chimeric proteins, any cofactors and any modules,combinations or fragments thereof.

Cell-Based Assays

In some embodiments, the present invention provides cell-based assays.As used herein, the term “cell-based assay” refers to an assaycomprising at least one aspect that involves the use of a living cell orcell culture. In some embodiments, these may be useful for assessing themodulation of growth factor release from GPCs, referred to herein as“growth factor release assays”. In some embodiments, cell-based assaysmay be useful for assessing the modulation of growth factor activity,referred to herein as “growth factor activity assays”. Cell-based assaysof the present invention may comprise expression cells and/or responsivecells. Expression cells, as referred to herein, are cells that expressone or more factors being analyzed in a particular assay. Suchexpression may be natural or may be the result of transfection and/ortransduction of a foreign gene. In some embodiments, expression of oneor more factors by expression cells may be enhanced or suppressed by theaddition of one or more exogenous factors. In some embodiments,expression cells may comprise cell lines (e.g. HEK293 cells, CHO cells,TMLC cells, 293T/17 cells, Hs68 cells, CCD1112sk cells, HFF-1 cells,Keloid fibroblasts or Sw-480 cells.) In some embodiments, cell linescomprising expression cells may express one or more recombinant proteinsof the present invention (e.g. naturally and/or through transfection,stable transfection, and/or transduction).

In some embodiments, growth factor release/activity assays may compriseexpression cells that express GPCs. In such embodiments, additionalfactors may be co-expressed in and/or combined with expression cells todetermine their effect on growth factor release from such GPCs. In someembodiments, integrins (including, but not limited to 46 integrin,α_(v)β₈ integrin and/or α₉β₁ integrin) are co-expressed and/or otherwiseintroduced to GPC-expressing expression cells. In some embodiments, suchadditional integrin expression may facilitate growth factor release. Insome embodiments, LTBPs, fibrillins and/or GARPs and/or variants thereofare coexpressed and/or otherwise introduced into expression cells.

In some embodiments, one or more genes may be knocked out, knocked downand/or otherwise modulated in expression cells depending on the focus ofa particular assay. In some embodiments, one or more gene products maybe modulated at the RNA and/or protein level. In some embodiments, geneproducts may be reduced through the introduction of siRNA molecules toexpression cells. In some embodiments, gene products from LTBP,fibrillin and/or GARP genes may be reduced and/or eliminated fromexpression cells of the present invention.

Cell-based assays of the present invention, including, but not limitedto growth factor release/activity assays, may comprise responsive cells.As used herein, the term “responsive cell” refers to a cell thatundergoes a response to one or more factors introduced into an assay. Insome embodiments, such responses may include a change in geneexpression, wherein such cells modulate transcription of one or moregenes upon contact with one or more factors introduced. In someembodiments, responsive cells may undergo a change in phenotype,behavior and/or viability.

In some embodiments, responsive cells comprise one or more reportergenes. As used herein, the term “reporter gene” refers to a syntheticgene typically comprising a promoter and a protein coding regionencoding one or more detectable gene products. Reporter genes aretypically designed in a way such that their expression may be modulatedin response to one or more factors being analyzed by a particular assay.This may be carried out by manipulating the promoter of reporter genes.As used herein, the term promoter refers to part of a gene thatinitiates transcription of that gene. Promoters typically comprisenucleotides at the 3′ end of the antisense strand of a given gene andare not transcribed during gene expression. Promoters typically functionthrough interaction with one or more transcription factors as well asRNA polymerase enzymes to initiate transcription of the protein encodingportion of the gene. Segments of the promoter that physically interactwith one or more transcription factors and/or polymerase enzymes arereferred to herein as response elements. In some embodiments, reportergenes are designed to comprise promoters and/or response elements knownto be responsive to one or more factors (including, but not limited togrowth factors) being analyzed in a given assay. Changes in responsivecell gene expression may be measured according to any methods availablein the art to yield gene expression data. Such gene expression data maybe obtained in the form of luciferase activity data [often measured interms of relative light units (RLUs.)]

In some cases, responsive cells undergo a change in viability inresponse to one or more factors introduced in an assay. Such responsivecells may be used in proliferation assays as described herein. Changesin responsive cell viability may be detected by cell counting and/orother methods known to those skilled the art to yield responsive cellviability data.

Protein encoding regions of reporter genes typically encode one or moredetectable proteins. Detectable proteins refer to any proteins capableof detection through one or more methods known in the art. Suchdetection methods may include, but are not limited to Western blotting,ELISA, assaying for enzymatic activity of detectable proteins (e.g.catalase activity, β-galactosidase activity and/or luciferase activity)immunocytochemical detection, surface plasmon resonance detection and/ordetection of fluorescent detectable proteins. When a reporter gene isused in an assay, the expression of detectable proteins correlates withthe ability of factors being assayed to activate the promoter present inthe reporter gene. In embodiments comprising growth factorrelease/activity assays, reporter gene promoters typically respond togrowth factor signaling. In such embodiments, the level of detectableprotein produced correlates with level of growth factor signaling,indicating release and/or activity of a given growth factor.

In some embodiments, reporter genes encode luciferase enzymes. Chemicalreactions between luciferase enzymes and substrate molecules arelight-emitting reactions. Due to such light-emitting reactions,luciferase enzyme levels can be quantified through the addition ofsubstrate molecules and subsequent photodetection of the emitted light.In some embodiments, reporter genes of the present invention encodefirefly luciferase, the sequence of which was cloned from Photinuspyralis. In some embodiments, responsive cells of the present inventioncomprise reporter genes that express luciferase with promoters that areresponsive to growth factors. In such embodiments, luciferase activitymay correlate with growth factor activity levels allowing for growthfactor activity and/or release from GPCs to be determined.

In some embodiments, reporter genes are inserted into bacterial plasmidsto enable replication and/or facilitate introduction into cells. In someembodiments, such plasmids are designed to comprise sequences encodingdetectable gene products and may be manipulated to insert promotersequences that may be responsive to one or more factors of interest.These plasmids are referred to herein as reporter plasmids. In someembodiments of the present invention, promoters that may be responsiveto one or more factors of interest may be inserted into reporterplasmids, upstream of sequences encoding detectable gene products toform functional reporter genes within such reporter plasmids. Reporterplasmids that comprise at least one functional reporter gene arereferred to herein as reporter constructs. In some embodiments, reporterconstructs of the present invention may comprise pGL2 reporter plasmids(Promega BioSciences, LLC, Madison, Wis.), pGL3 reporter plasmids(Promega BioSciences, LLC, Madison, Wis.), pGL4 reporter plasmids(Promega BioSciences, LLC, Madison, Wis.) or variants thereof. Suchreporter constructs express firefly luciferase in response to promoteractivation.

In some embodiments, reporter constructs may be introduced directly intoexpression cells or may be introduced into one or more responsive cells.Responsive cells of the present invention comprising one or morereporter genes are referred to herein as reporter cells. In someembodiments, reporter cells may be transiently transfected with reporterconstructs or may comprise stable expression of such constructs (e.g.reporter constructs are successfully replicated along with genomic DNAduring each round of cell division). Cell lines that stably comprisereporter constructs are referred to herein as reporter cell lines. Insome embodiments, reporter cells are mammalian. In some embodiments,reporter cells may comprise mouse cells, rabbit cells, rat cells, monkeycells, hamster cells and human cells. In some embodiments, cell linesuseful for transient and/or stable expression of reporter genes mayinclude, but are not limited to HEK293 cells, HeLa cells, Sw-480 cells,TMLC cells [as disclosed by Abe et al (Abe, M. et al., An assay fortransforming growth factor-β using cells transfected with a plasminogenactivator inhibitor-1 promoter-luciferase construct. AnalyticalBiochemistry. 1994. 216:276-84)] 293T/17 cells, Hs68 cells, CCD1112skcells, HFF-1 cells, Keloid fibroblasts, A204 cells, L17 RIB cells [asdisclosed by Cash et al (Cash, J. N et al., The structure ofmyostatin:follistatin 288: insights into receptor utilization andheparin binding. The EMBO Journal. 2009. 28:2662-76)] C₂C₁₂ cells andEL4 T lymphoma cells.

In embodiments where one or more reporter cells and/or reporter celllines are utilized, such cells may be cultured with expression cells aspart of a co-culture system. In some embodiments reporter cells/reportercell lines may be cultured separately from expression cells. In suchembodiments, lysates and/or media from expression cells may be combinedwith reporter cell/reporter cell line cultures to assess expressedfactors (including, but not limited to growth factors).

In some embodiments, cell-based assays of the present invention may onlycomprise expression cells and not responsive cells. In such embodiments,expressed proteins, including but not limited to GPCs and/or growthfactors, may be detected by one or more methods that are not cell based.Such methods may include, but are not limited to Western Blotting,enzyme-linked immunosorbent assay (ELISA) immunocytochemistry, surfaceplasmon resonance and other methods known in the art for proteindetection. In some embodiments, TGF-β release in expression cellcultures and/or culture medium may be detected by ELISA. In someembodiments, such assays may utilize anti-TGF-β antibody, clone 1D11antibody (R&D Systems, Minneapolis, Minn.) as a capture antibody,capable of recognizing TGF-β isoforms 1, 2 and 3 in multiple species,including, but not limited to cows, chickens, mice and humans. In someembodiments, biotinylated anti-TGF-β1 chicken IgY (BAF240; R&D Systems,Minneapolis, Minn.) may be used as a detection antibody. In someembodiments, GDF-8/myostatin release in expression cell cultures and/orculture medium may be detected by ELISA. In some embodiments, theGDF-8/myostatin quantikine ELISA kit (R&D Systems, Minneapolis, Minn.)may be used. Examples of anti-GDF-8/myostatin antibodies that may beused for detection include AF1539, MAB788 and AF788 (R&D Systems,Minneapolis, Minn.)

In some embodiments, reporter genes of the present invention comprisegrowth factor-responsive promoters. As used herein, the term “growthfactor-responsive promoter” refers to a gene promoter that facilitatestranscription of a downstream gene in response to growth factor cellsignaling induced by one or more growth factors. In some embodiments,growth factor-responsive promoters are responsive to TGF-β family membergrowth factor signaling. In some embodiments, growth factor-responsivepromoters of the present invention comprise one or more sequences listedin Table 16 or fragments or variants thereof. These include two versionsof the plasminogen activator inhibitor type 1 (PAI-1) promoter [V1 asdisclosed by Abe et al (Abe, M. et al., An assay for transforming growthfactor-β using cells transfected with a plasminogen activatorinhibitor-1 promoter-luciferase construct. Analytical Biochemistry.1994. 216:276-84) and V2 as disclosed in WO 2011/034935, the contents ofwhich are hereby incorporated by reference in their entirety,] acollagen, type 1, alpha 1 promoter, a collagen, type 1, alpha 2promoter, a FoxP3 promoter, a CAGA12 promoter [responsive toSmad-dependent signaling as reporter by Thies et al (Thies, R. S. etal., GDF-8 propeptide binds to GDF-8 and antagonizes biological activityby inhibiting GDF-8 receptor binding. Growth Factors. 2001. 18:251-9)and an adenovirus major late promoter.

TABLE 16 Growth factor-responsive promoters SEQ ID Promoter Sequence NOPAI-I(V1) AGCTTACCATGGTAACCCCTGGTCCCGTTCAGCCACCACCACCC 258CACCCAGCACACCTCCAACCTCAGCCAGACAAGGTTGTTGACACAAGAGAGCCCTCAGGGGCACAGAGAGAGTCTGGACACGTGGGGAGTCAGCCGTGTATCATCGGAGGCGGCCGGGCACATGGCAGGGATGAGGGAAAGACCAAGAGTCCTCTGTTGGGCCCAAGTCCTAGACAGACAAAACCTAGACAATCACGTGGCTGGCTGCATGCCTGTGGCTGTTGGGCTGGGCAGGAGGAGGGAGGGGCGCTCTTTCCTGGAGGTGGTCCAGAGCACCGGGTGGACAGCCCTGGGGGAAAACTTCCACGTTTTGATGGAGGTTATCTTTGATAACTCCACAGTGACCTGGTTCGCCAAAGGAAAAGCAGGCAACGTGAGCTGTTTTTTTTTTCTCCAAGCTGAACACTAGGGGTCCTAGGCTTTTTGGGTCACCCGGCATGGCAGACAGTCAACCTGGCAGGACATCCGGGAGAGACAGACACAGGCAGAGGGCAGAAAGGTCAAGGGAGGTTCTCAGGCCAAGGCTATTGGGGTTTGCTCAATTGTTCCTGAATGCTCTTACACACGTACACACACAGAGCAGCACACACACACACACACACATGCCTCAGCAAGTCCCAGAGAGGGAGGTGTCGAGGGGGACCCGCTGGCTGTTCAGACGGACTCCCAGAGCCAGTGAGTGGGTGGGGCTGGAACATGAGTTCATCTATTTCCTGCCCACATCTGGTATAAAAGGAGGCAGTGGCCCACAGAGGAGCACAGCTGTGTTTGGCTGCAGGGCCAAGAGCGCTGTCAAGAAGACCCACACGCCCCCC TCCAGCAGCTG PAI-1(V2)TTGGTCTCCTGTTTCCTTACCAAGCTTTTACCATGGTAACCCCTG 259GTCCCGTTCAGCCACCACCACCCCACCCAGCACACCTCCAACCTCAGCCAGACAAGGTTGTTGACACAAGAGAGCCCTCAGGGGCACAGAGAGAGTCTGGACACGTGGGGAGTCAGCCGTGTATCATCGGAGGCGGCCGGGCACATGGCAGGGATGAGGGAAAGACCAAGAGTCCTCTGTTGGGCCCAAGTCCTAGACAGACAAAACCTAGACAATCACGTGGCTGGCTGCATGCCCTGTGGCTGTTGGGCTGGGCCCAGGAGGAGGGAGGGGCGCTCTTTCCTGGAGGTGGTCCAGAGCACCGGGTGGACAGCCCTGGGGGAAAACTTCCACGTTTTGATGGAGGTTATCTTTGATAACTCCACAGTGACCTGGTTCGCCAAAGGAAAAGCAGGCAACGTGAGCTGTTTTTTTTTTCTCCAAGCTGAACACTAGGGGTCCTAGGCTTTTTGGGTCACCCGGCATGGCAGACAGTCAACCTGGCAGGACATCCGGGAGAGACAGACACAGGCAGAGGGCAGAAAGGTCAAGGGAGGTTCTCAGGCCAAGGCTATTGGGGTTTGCTCAATTGTTCCTGAATGCTCTTACACACGTACACACACAGAGCAGCACACACACACACACACACATGCCTCAGCAAGTCCCAGAGAGGGAGGTGTCGAGGGGGACCCGCTGGCTGTTCAGACGGACTCCCAGAGCCAGTGAGTGGGTGGGGCTGGAACATGAGTTCATCTATTTCCTGCCCACATCTGGTATAAAAGGAGGCAGTGGCCCACAGAGGAGCACAGCTGTGTTTGGCTGCAGGGCCAAGAGCGCTGTCAAGAAGACCCACACGCCCCCCTCCAGCAGCTGAATTCCTGCAGCTCAGCAGCCGCCGCCAGAGCAGGACGAACCGCCAATCGC AAGGCACCTCTGAGAACTTCAGGTACol1A1 CCATGGCAAACAAAACTCTTCTCTAAGTCACCAATGATCACAG 260GCCTCCCACTAAAAATACTTCCCAACTCTGGGGTGGAAGAGTTTGGGGGATGAATTTTTAGGGGATTGCAAGCCCCAATCCCCACCTCTGTGTCCCTAGAATCCCCCACCCCTACCTTGGCTGCTCCATCACCCAACCACCAAAGCTTTCTTCTGCAGAGGCCACCTAGTCATGTTTCTCACCCTGCACCTCAGCCTCCCCACTCCATCTCTCAATCATGCCTAGGGTTTGGAGGAAGGCATTTGATTCTGTTCTGGAGCACAGCAGAAGAATTGACATCCTCAAAATTAAAACTCCCTTGCCTGCACCCCTCCCTCAGATATCTGATTCTTAATGTCTAGAAAGGAATCTGTAAATTGTTCCCCAAATATTCCTAAGCTCCATCCCCTAGCCACACCAGAAGACACCCCCAAACAGGCACATCTTTTTAATTCCCAGCTTCCTCTGTTTTGGAGAGGTCCTCAGCATGCCTCTTTATGCCCCTCCCTTAGCTCTTGCCAGGATATCAGAGGGTGACTGGGGCACAGCCAGGAGGACCCCCTCCCCAACACCCCCAACCCTTCCACCTTTGGAAGTCTCCCCACCCAGCTCCCCAGTTCCCCAGTTCCACTTCTTCTAGATTGGAGGTCCCAGGAAGAGAGCAGAGGGGCACCCCTACCCACTGGTTAGCCCACGCCATTCTGAGGACCCAGCTGCACCCCTACCACAGCACCTCTGGCCCAGGCTGGGCTGGGGGGCTGGGGAGGCAGAGCTGCGAAGAGGGGAGATGTGGGGTGGACTCCCTTCCCTCCTCCTCCCCCTCTCCATTCCAACTCCCAAATTGGGGGCCGGGCCAGGCAGCTCTGATTGGCTGGGGCACGGGCGGCCGGCTCCCCCTCTCCGAGGGGCAGGGTTCCTCCCTGCTCTCCATCAGGACAGTATAAAAGGGGCCCGGGCCAGTCGTCGGAGCAGACGGGAGTTTCTCCTCGGGGTCGGAGCAGGAGGCACGCGGAGTGTGAGGCCACGCATGAGCGGACGCTAACCCCCTCCCCAGCCACAAA GAGTCTACATG Col1A2TAGAGTTCGCAAAGCCTATCCTCCCTGTAGCCGGGTGCCAAGC 261AGCCTCGAGCCTGCTCCCCAGCCCACCTGCCAACAAAAGGCGCCCTCCGACTGCAACCCAGCCCTCCACAGACAGGACCCGCCCTTTCCCGAAGTCATAAGACAAAGAGAGTGCATCACTGCTGAAACAGTGGGCGCACACGAGCCCCAAAGCTAGAGAAAAGCTGGACGGGGCTGGGGGCGGGGTGCAGGGGTGGAGGGGCGGGGAGGCGGGCTCCGGCTGCGCCACGCTATCGAGTCTTCCCTCCCTCCTTCTCTGCCCCCTCCGCTCCCGCTGGAGCCCTCCACCCTACAAGTGGCCTACAGGGCACAGGTGAGGCGGGACTGGACAGCTCCTGCTTTGATCGCCGGAGATCTGCAAATTCTGCCCATGTCGGGGCTGCAGAGCACTCCGACGTGTCCCATAGTGTTTCCAAACTTGGAAAGGGCGGGGGAGGGCGGGAGGATGCGGAGGGCGGAGGTATGCAGACAACGAGTCAGAGTTTCCCCTTGAAAGCCTCAAAAGTGTCCACGTCCTCAAAAAGAATGGAACCAATTTAAGAAGCCAGCCCCGTGGCCACGTCCCTTCCCCCATTCGCTCCCTCCTCTGCGCCCCCGCAGGCTCCTCCCAGCTGTGGCTGCCCGGGCCCCCAGCCCCAGCCCTCCCATTGGTGGAGGCCCTTTTGGAGGCACCCTAGGGCCAGGGAAACTTTTGCCGTATAAATAGGGCAGATCCGGGCTTTATTATTTTAGCACCACGGCAGCAGGAGGTTTCGGCTAAGTTGGAGGTACTGGCCACGACTGCATGCCCGCGCCCGCCAGGTGATACCTCCGCCGGTGACCCAGGGGCTCTGCGACACAAGGAGTCTGCATGTCTAAGTGCTAGACATGCTCAGCTTTGTGGATACGCGGACTTTGTTGCTGCTTGC AGTAA FoxP3AGTAAAAGACCCCAAAGGCTGAGGGCCTCAGAAGCATCAGGC 262CATGATGTTCCTGAAACAAGAGGGTCAGGGTCCCAATGGGCCTCTGGGGTTCATCGTGAGGATGGATGCATTAATATTGGGGACCTGCTAGGGACCTTCCCAGTGGGACAGTGGCTGGGTCAGGGCACTCAAGCCCTAAAACGTGATGAGGCGAGACTTTTCTCTCTTTCCTCATTCAGTAACTGTCAGTAGATTCTGGGAGCCAGGGATTCTCCGACTCTTCAAGTCCATGAATTTTAGGGGATGACAGTGGGCTCTCCGCTTTCTCCTCCATGAAGTAACTTACATGCCCCTCACCCTCTGTGGGAGGGGTGTTGCAGGGGGTGCAGAACTCCCCTCGCCGGGTAGTTCAAGCAATGGGGACCATATCAATTCCATCTATAGGGAAACTGAGGCCTGGAGTAGGGCGAGGCCTCTGGGAACCCAGCCCTATTCTGTCTCTTTCCCTGGCATTTCCCATCCACACATAGAGCTTCAGATTCTCTTTCTTTCCCCAGAGACCCTCAAATATCCTCTCACTCACAGAATGGTGTCTCTGCCTGCCTCGGGTTGGCCCTGTGATTTATTTTAGTTCTTTTCCCTTGTTTTTTTTTTTTCAAACTCTATACACTTTTGTTTTAAAAACTGTGGTTTCTCATGAGCCCTATTATCTCATTGATACCTCTCACCTCTGTGGTGAGGGGAAGAAATCATATTTTCAGATGACTCGTAAAGGGCAAAGAAAAAAACCCAAAATTTCAAAATTTCCGTTTAAGTCTCATAATCAAGAAAAGGAGAAACACAGAGAGAGAGAAAAAAAAAACTATGAGAACCCCCCCCCACCCCGTGATTATCAGCGCACACACTCATCGAAAAAAATTTGGATTATTAGAAGAGAGAGGTCTGCGGCTTCCACACCGTACAGCGTGGTTTTTCTTCTCGGTATAAAAGCAAAGTTGTTTTTGATACGTGACAGTTTCCCACAAGCCAGGCTGATCCTTTTCTGTCAGTCCACTTCACCA CAGA12AGCCAGACAAGCCAGACAAGCCAGACAAGCCAGACAAGCCAG 263ACAAGCCAGACAAGCCAGACAAGCCAGACAAGCCAGACAAGC CAGACAAGCCAGACAAGCCAGACAAdenovirus GGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTCTT 264 major lateCCG promoter

In some embodiments, mink lung epithelial/PAI reporter cell lines may beused. Mink lung epithelial cells do not produce TGF-β, but do expresshigh levels of TGF-β receptors (Munger et al.) Mink lung epithelial/PAIreporter cell lines comprise reporter constructs comprising promoterelements from the TGF-β-responsive genes PAI and/or COL1A that modulatethe expression of the protein coding portion of the luciferase gene. Insome embodiments, other reporter constructs may be used with mink lungepithelial cells. In some embodiments, SMAD3-responsive reporterconstructs may be used.

TGF-β2 Release Assay

In some embodiments, the present invention provides assays for detectingthe release and/or activity of TGF-β2. Such assays may comprise celllines (e.g. HEK293 cells, 293T/17 cells, Hs68 cells, CCD1112sk cells,HFF-1 cells, Keloid fibroblasts or Sw-480 cells) that express GPCscomprising TGF-β2 (e.g. naturally and/or through transfection, stabletransfection, and/or transduction) and/or recombinant and/or chimericprotein derivatives thereof. In some embodiments, additional factors areexpressed in and/or combined with TGF-β2-expressing cells to determinetheir effect on TGF-β2 growth factor release. In some embodiments,integrins may be expressed. In some embodiments, α₉β₁ integrin may beexpressed.

In some embodiments, TGF-β2 release may be detected by one or moregrowth factor release assays according to those described herein. Insome embodiments, such assays may comprise the use of mink lungepithelial/PAI reporter cell lines to measure TGF-β2 release and/oractivity. In some embodiments, TGF-β2 release assays may be used toscreen antibodies for inhibitory and/or activating properties withregard to TGF-β2 release from GPCs and/or activity

T_(reg) Induction Assay

T_(reg) cells are immune cells that comprise a suppressor cell functionimportant in regulating autoimmunity. Such cells are derived fromprecursor cells after the induction of the FoxP3 gene (Wood andSakaguchi, Nature Reviews, 2003). FoxP3 is a transcription factor, theexpression of which may be regulated to some degree by TGF-β-relatedproteins. Wan and Flavell (2005) demonstrated that in response toexogenous TGF-β, activated primary T cells show de novo FoxP3 and“knocked-in” fluorescent protein expression and induction of suppressorcell function. Tone et al (2008) demonstrated that key TGF-β responsiveenhancer elements that drive FoxP3 expression in primary T cells arepresent in the EL4 T lymphoma line. In some embodiments, the presentinvention provides reporter constructs comprising promoter elements fromthe FoxP3 gene that modulate expression of such reporter constructs(referred to herein as FoxP3-driven reporter constructs). In someembodiments, FoxP3-driven reporter constructs comprise promoter elementsresponsive to TGF-β-related protein cell signaling activity. In someembodiments, FoxP3-driven reporter constructs are introduced(transiently and/or stably) to one or more cells and/or cell lines. Suchcells are referred to herein as FoxP3-driven reporter cells. In someembodiments, such cells are mammalian. In some embodiments, suchmammalian cells may include, but are not limited to mouse cells, rabbitcells, rat cells, monkey cells, hamster cells and human cells. Suchcells may be derived from a cell line. In some embodiments, human cellsmay be used. In some embodiments, cell lines may include, but are notlimited to HEK293 cells, HeLa cells, Sw-480 cells, EL4 T lymphoma cells,TMLC cells, 293T/17 cells, Hs68 cells, CCD1112sk cells, HFF-1 cells,Keloid fibroblasts, A204 cells, L17 RIB cells and C₂C₁₂ cells. In someembodiments, EL4 T lymphoma cells may be used. EL4 T lymphoma cells areknown to comprise transcriptional enhancer elements that are responsiveto TGF-β-related protein signaling. In some embodiments, FoxP3-drivenreporter cells may be used to screen antibodies for their ability toactivate and/or inhibit FoxP3-dependent gene expression.

Proliferation Assays

In some embodiments, cell-based assays of the present invention maycomprise proliferation assays. As used herein, the term “proliferationassay” refers to an assay that determines the effect on one or moreagents on cell proliferation.

In some cases, proliferation assays may comprise HT2 proliferationassays. Such assays may be carried out, for example, according to themethods described in Tsang, M. et al., 1995. Cytokine 7(5):389-97, thecontents of which are herein incorporated by reference in theirentirety. HT2 cells (ATCC CRL-1841) are grown in the presence of IL-2,in which they are insensitive to TGF-β1 in the culture media. When HT2cells are switched into IL-4-containing media they will continue toproliferate, but will respond to TGF-β1 in the culture media byinduction of apoptosis. In IL-4 containing media, cell death due toTGF-β1 in culture media occurs in a dose dependent manner, which can beblocked by numerous reagents interfering with the TGF-β signalingpathway. This enables the use of this assay to screen reagents tomodulate TGF-β1 activation.

Detection of changes in cell number may be carried out, in someembodiments, through the detection and/or quantification of ATP levelsin cells. ATP levels typically correlate with the number of cellspresent in a given test sample, well, plate or dish. In someembodiments, ATP levels may be determined using a CELLTITER-GLO®Luminescent Cell Viability Assay (Promega BioSciences, LLC, Madison,Wis.).

Kits and Devices

Any of the compounds and/or compositions of the present invention may becomprised in a kit. In a non-limiting example, reagents for generatingcompounds and/or compositions, including antigen molecules are includedin one or more kit. In some embodiments, kits may further includereagents and/or instructions for creating and/or synthesizing compoundsand/or compositions of the present invention. In some embodiments, kitsmay also include one or more buffers. In some embodiments, kits of theinvention may include components for making protein or nucleic acidarrays or libraries and thus, may include, for example, solid supports.

In some embodiments, kit components may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquotted. Where there are more than one kitcomponent, (labeling reagent and label may be packaged together), kitsmay also generally contain second, third or other additional containersinto which additional components may be separately placed. In someembodiments, kits may also comprise second container means forcontaining sterile, pharmaceutically acceptable buffers and/or otherdiluents. In some embodiments, various combinations of components may becomprised in one or more vial. Kits of the present invention may alsotypically include means for containing compounds and/or compositions ofthe present invention, e.g., proteins, nucleic acids, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich desired vials are retained.

In some embodiments, kit components are provided in one and/or moreliquid solutions. In some embodiments, liquid solutions are aqueoussolutions, with sterile aqueous solutions being particularly preferred.In some embodiments, kit components may be provided as dried powder(s).When reagents and/or components are provided as dry powders, suchpowders may be reconstituted by the addition of suitable volumes ofsolvent. In some embodiments, it is envisioned that solvents may also beprovided in another container means. In some embodiments, labeling dyesare provided as dried powders. In some embodiments, it is contemplatedthat 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150,160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000micrograms or at least or at most those amounts of dried dye areprovided in kits of the invention. In such embodiments, dye may then beresuspended in any suitable solvent, such as DMSO.

In some embodiments, kits may include instructions for employing kitcomponents as well the use of any other reagent not included in the kit.Instructions may include variations that may be implemented.

In some embodiments, compounds and/or compositions of the presentinvention may be combined with, coated onto or embedded in a device.Devices may include, but are not limited to, dental implants, stents,bone replacements, artificial joints, valves, pacemakers and/or otherimplantable therapeutic device.

Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. The following is a non-limiting list of term definitions.

Activity: As used herein, the term “activity” refers to the condition inwhich things are happening or being done. Compositions of the inventionmay have activity and this activity may involve one or more biologicalevents. In some embodiments, such biological event may involve growthfactors and/or growth factor signaling. In some embodiments, biologicalevents may include cell signaling events associated with growth factorand receptor interactions. In some embodiments, biological events mayinclude cell signaling events associated with TGF-β or TGF-β-relatedprotein interactions with one or more corresponding receptors.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” refers to simultaneousexposure of one or more subjects to two or more agents administered atthe same time or within an interval such that the subject is at somepoint in time simultaneously exposed to both and/or such that there maybe an overlap in the effect of each agent on the patient. In someembodiments, at least one dose of one or more agents is administeredwithin about 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, 30 minutes,15 minutes, 10 minutes, 5 minutes, or 1 minute of at least one dose ofone or more other agents. In some embodiments, administration occurs inoverlapping dosage regimens. As used herein, the term “dosage regimen”refers to a plurality of doses spaced apart in time. Such doses mayoccur at regular intervals or may include one or more hiatus inadministration. In some embodiments, the administration of individualdoses of one or more compounds and/or compositions of the presentinvention, as described herein, are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” refers to those proteinsand/or other biomolecules provided herein that are immunospecificallybound or interact with antibodies of the present invention and/orfragments, mutants, variants, and/or alterations thereof describedherein. In some embodiments, antigens of interest may compriseTGF-β-related proteins, growth factors, prodomains, GPCs, proteinmodules or regions of overlap between them.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, mean that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serve as linking agents, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Biomolecule: As used herein, the term “biomolecule” is any naturalmolecule which is amino acid-based, nucleic acid-based,carbohydrate-based or lipid-based, and the like.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, acompounds and/or compositions of the present invention may be consideredbiologically active if even a portion of is biologically active ormimics an activity considered to biologically relevant.

Biological system: As used herein, the term “biological system” refersto a group of organs, tissues, cells, intracellular components,proteins, nucleic acids, molecules (including, but not limited tobiomolecules) that function together to perform a certain biologicaltask within cellular membranes, cellular compartments, cells, tissues,organs, organ systems, multicellular organisms, or any biologicalentity. In some embodiments, biological systems are cell signalingpathways comprising intracellular and/or extracellular cell signalingbiomolecules. In some embodiments, biological systems comprise growthfactor signaling events within the extracellular matrix, cellular matrixand/or cellular niches.

Candidate antibody: As used herein, the term “candidate antibody” refersto an antibody from a pool of one or more antibody from which one ormore desired antibodies may be selected.

Cellular matrix: As used herein, the term “cellular matrix” refers tothe biochemical and structural environment associated with the outerportion of the cell membrane. Such cell membranes may also includeplatelet membranes. Components of the cellular matrix may include, butare not limited to proteoglycans, carbohydrate molecules, integralmembrane proteins, glycolipids and the like. In some cases, cellularmatrix components may include growth factors and/or modulators of growthfactor activity. Some cellular matrix proteins include integrins, GARPand LRRC33.

Compound: As used herein, the term “compound,” refers to a distinctchemical entity The term may be used herein to refer to peptides,proteins, protein complexes or antibodies of the invention. In someembodiments, a particular compound may exist in one or more isomeric orisotopic forms (including, but not limited to stereoisomers, geometricisomers and isotopes). In some embodiments, a compound is provided orutilized in only a single such form. In some embodiments, a compound isprovided or utilized as a mixture of two or more such forms (including,but not limited to a racemic mixture of stereoisomers). Those of skillin the art appreciate that some compounds exist in different such forms,show different properties and/or activities (including, but not limitedto biological activities). In such cases it is within the ordinary skillof those in the art to select or avoid particular forms of the compoundfor use in accordance with the present invention. For example, compoundsthat contain asymmetrically substituted carbon atoms can be isolated inoptically active or racemic forms. Methods on how to prepare opticallyactive forms from optically active starting materials are known in theart, such as by resolution of racemic mixtures or by stereoselectivesynthesis.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of polynucleotide or polypeptide sequences,respectively, that are those that occur unaltered in the same positionof two or more sequences being compared. Nucleotides or amino acids thatare relatively conserved are those that are conserved among more relatedsequences than nucleotides or amino acids appearing elsewhere in thesequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

In one embodiment, conserved sequences are not contiguous. Those skilledin the art are able to appreciate how to achieve alignment when gaps incontiguous alignment are present between sequences, and to aligncorresponding residues not withstanding insertions or deletions present.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any agentwhich facilitates, at least in part, the in vivo delivery of one or moresubstances (including, but not limited to a compounds and/orcompositions of the present invention) to a cell, subject or otherbiological system cells.

Desired antibody: As used herein, the term “desired antibody” refers toan antibody that is sought after, in some cases from a pool of candidateantibodies.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, reference, wild-type or native form of the same regionor molecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity, which markers, signals or moieties arereadily detected by methods known in the art including radiography,fluorescence, chemiluminescence, enzymatic activity, absorbance,immunological detection and the like. Detectable labels may includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands, biotin, avidin, streptavidin and haptens, quantum dots,polyhistidine tags, myc tags, flag tags, human influenza hemagglutinin(HA) tags and the like. Detectable labels may be located at any positionin the entity with which they are attached, incorporated or associated.For example, when attached, incorporated in or associated with a peptideor protein, they may be within the amino acids, the peptides, orproteins, or located at the N- or C-termini.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule. Thus, engineered agents or entities are thosewhose design and/or production include an act of the hand of man.

Epitope: As used herein, an “epitope” refers to a surface or region on amolecule that is capable of interacting with components of the immunesystem, including, but not limited to antibodies. In some embodiments,when referring to a protein or protein module, an epitope may comprise alinear stretch of amino acids or a three dimensional structure formed byfolded amino acid chains.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; (4) folding of a polypeptide or protein; and (5)post-translational modification of a polypeptide or protein.

Extracellular matrix: As used herein, the term, “extracellular matrix,”or “ECM” refers to the area surrounding cells and/or the area betweencells that typically comprises structural proteins as well as cellsignaling molecules. Components of the extracellular matrix may include,but are not limited to proteins, nucleic acids, membranes, lipids andsugars that may be directly or indirectly associated with structuralcomponents of the extracellular environments. Structural components ofthe extracellular matrix may include, but are not limited to proteins,polysaccharides (e.g. hyaluronic acid) glycosaminoglycans andproteoglycans (e.g. heparin sulfate, chondroitin sulfate and keratinsulfate.) Such structural components may include, but are not limited tofibrous components (e.g. collagens and elastins) fibrillins,fibronectin, laminins, agrin, perlecan, decorin and the like. Otherproteins that may be components of the extracellular matrix include andLTBPs. Extracellular matrix components may also include growth factorsand/or modulators of growth factor activity.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least acompound and/or composition of the present invention and a deliveryagent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells. In someembodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more aminoacids. In some embodiments, fragments of an antibody include portions ofan antibody subjected to enzymatic digestion or synthesized as such.

Functional: As used herein, a “functional” biological molecule is abiological entity with a structure and in a form in which it exhibits aproperty and/or activity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is typically determined by theability to encode a stretch of at least 4-5 uniquely specified aminoacids. In accordance with the invention, two protein sequences areconsidered to be homologous if the proteins are at least about 50%, 60%,70%, 80%, or 90% identical for at least one stretch of at least about 20amino acids. In many embodiments, homologous protein may show a largeoverall degree of homology and a high degree of homology over at leastone short stretch of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acids. Inmany embodiments, homologous proteins share one or more characteristicsequence elements. As used herein, the term “characteristic sequenceelement” refers to a motif present in related proteins. In someembodiments, the presence of such motifs correlates with a particularactivity (such as biological activity).

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, may be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determined, forexample using the algorithm of Meyers and Miller (CABIOS, 1989,4:11-17), which has been incorporated into the ALIGN program (version2.0) using a PAM120 weight residue table, a gap length penalty of 12 anda gap penalty of 4. The percent identity between two nucleotidesequences can, alternatively, be determined using the GAP program in theGCG software package using an NWSgapdna.CMP matrix. Methods commonlyemployed to determine percent identity between sequences include, butare not limited to those disclosed in Carillo, H., and Lipman, D., SIAMJ Applied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product may be RNAtranscribed from the gene (e.g. mRNA) or a polypeptide translated frommRNA transcribed from the gene. Typically a reduction in the level ofmRNA results in a reduction in the level of a polypeptide translatedtherefrom. The level of expression may be determined using standardtechniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” is synonymous with“separated”, but carries with it the inference separation was carriedout by the hand of man. In one embodiment, an isolated substance orentity is one that has been separated from at least some of thecomponents with which it was previously associated (whether in nature orin an experimental setting). Isolated substances may have varying levelsof purity in reference to the substances from which they have beenassociated. Isolated substances and/or entities may be separated from atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, or more of the other components withwhich they were initially associated. In some embodiments, isolatedagents are more than about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or more than about 99% pure. As used herein, a substance is“pure” if it is substantially free of other components.

Substantially isolated: By “substantially isolated” is meant that thecompound is substantially separated from the environment in which it wasformed or detected. Partial separation can include, for example, acomposition enriched in the compound of the present disclosure.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compound of the present disclosure, or saltthereof. Methods for isolating compounds and their salts are routine inthe art. In some embodiments, isolation of a substance or entityincludes disruption of chemical associations and/or bonds. In someembodiments, isolation includes only the separation from components withwhich the isolated substance or entity was previously combined and doesnot include such disruption.

Linker: As used herein, a linker refers to a moiety that connects two ormore domains, moieties or entities. In one embodiment, a linker maycomprise 10 or more atoms. In a further embodiment, a linker maycomprise a group of atoms, e.g., 10-1,000 atoms, and can be comprised ofthe atoms or groups such as, but not limited to, carbon, amino,alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. Insome embodiments, a linker may comprise one or more nucleic acidscomprising one or more nucleotides. In some embodiments, the linker maycomprise an amino acid, peptide, polypeptide or protein. In someembodiments, a moiety bound by a linker may include, but is not limitedto an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase,a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, aprotein, a protein complex, a payload (e.g., a therapeutic agent). or amarker (including, but not limited to a chemical, fluorescent,radioactive or bioluminescent marker). The linker can be used for anyuseful purpose, such as to form multimers or conjugates, as well as toadminister a payload, as described herein. Examples of chemical groupsthat can be incorporated into the linker include, but are not limitedto, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester,alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can beoptionally substituted, as described herein. Examples of linkersinclude, but are not limited to, unsaturated alkanes, polyethyleneglycols (e.g., ethylene or propylene glycol monomeric units, e.g.,diethylene glycol, dipropylene glycol, triethylene glycol, tripropyleneglycol, tetraethylene glycol, or tetraethylene glycol), and dextranpolymers, Other examples include, but are not limited to, cleavablemoieties within the linker, such as, for example, a disulfide bond(—S—S—) or an azo bond (—N═N—), which can be cleaved using a reducingagent or photolysis. Non-limiting examples of a selectively cleavablebonds include an amido bond which may be cleaved for example by the useof tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond which may be cleaved forexample by acidic or basic hydrolysis.

Modified: As used herein, the term “modified” refers to a changed stateor structure of a molecule or entity as compared with a parent orreference molecule or entity. Molecules may be modified in many waysincluding chemically, structurally, and functionally. In someembodiments, compounds and/or compositions of the present invention aremodified by the introduction of non-natural amino acids.

Mutation: As used herein, the term “mutation” refers to a change and/oralteration. In some embodiments, mutations may be changes and/oralterations to proteins (including peptides and polypeptides) and/ornucleic acids (including polynucleic acids). In some embodiments,mutations comprise changes and/or alterations to a protein and/ornucleic acid sequence. Such changes and/or alterations may comprise theaddition, substitution and or deletion of one or more amino acids (inthe case of proteins and/or peptides) and/or nucleotides (in the case ofnucleic acids and or polynucleic acids). In embodiments whereinmutations comprise the addition and/or substitution of amino acidsand/or nucleotides, such additions and/or substitutions may comprise 1or more amino acid and/or nucleotide residues and may include modifiedamino acids and/or nucleotides.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid, or involvement of the hand ofman.

Niche: As used herein, the term “niche” refers to a place, zone and/orhabbitat. In some embodiments, niches comprise cellular niches. As usedherein, the term “cell niche” refers to a unique set of physiologicconditions in a cellular system within a tissue, organ or organ systemwithin or derived from a mammalian organism. A cell niche may occur invivo, in vitro, ex vivo, or in situ. Given the complex nature and thedynamic processes involved in growth factor signaling, a cell niche maybe characterized functionally, spatially or temporally or may be used torefer to any environment that encompasses one or more cells. As such, insome embodiments a cell niche includes the environment of any celladjacent to another cell that provides support, such as for example anurse cell. In some embodiments, niches may include those described inU.S. Provisional Patent Applications 61/722,919, filed Nov. 6, 2012 and61/722,969, filed Nov. 6, 2012, the contents of each of which are hereinincorporated by reference in their entireties.

Non-human vertebrate: As used herein, a “non-human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene and/or cellular transcript.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Passive adsorption: As used herein, “passive adsorption” refers to amethod of immobilizing solid-phase reactants on one or more surfaces(e.g. membranes, dishes, culture dishes, assay plates, etc.)Immobilization typically occurs due to affinity between such reactantsand surface components.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trained(e.g., licensed) professional for a particular disease or condition.

Peptide: As used herein, the term “peptide” refers to a chain of aminoacids that is less than or equal to about 50 amino acids long, e.g.,about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: As used herein, the term“pharmaceutically acceptable excipient,” as used herein, refers to anyingredient other than active agents (e.g., as described herein) presentin pharmaceutical compositions and having the properties of beingsubstantially nontoxic and non-inflammatory in subjects. In someembodiments, pharmaceutically acceptable excipients are vehicles capableof suspending and/or dissolving active agents. Excipients may include,for example: antiadherents, antioxidants, binders, coatings, compressionaids, disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspending or dispersing agents, sweeteners, and waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A,vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: Pharmaceutically acceptable salts ofthe compounds described herein are forms of the disclosed compoundswherein the acid or base moiety is in its salt form (e.g., as generatedby reacting a free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. Pharmaceutically acceptable salts include the conventionalnon-toxic salts, for example, from non-toxic inorganic or organic acids.In some embodiments a pharmaceutically acceptable salt is prepared froma parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety. Pharmaceuticallyacceptable solvate: The term “pharmaceutically acceptable solvate,” asused herein, refers to a crystalline form of a compound whereinmolecules of a suitable solvent are incorporated in the crystal lattice.For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.” In some embodiments, the solventincorporated into a solvate is of a type or at a level that isphysiologically tolerable to an organism to which the solvate isadministered (e.g., in a unit dosage form of a pharmaceuticalcomposition).

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to livingorganisms. Pharmacokinetics are divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may be covalently bonded or sequestered in some wayuntil converted into the active drug moiety prior to, upon or afteradministration to a mammalian subject. Prodrugs can be prepared bymodifying functional groups present in the compounds in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand, replicate or increase or cause to grow, expand, replicate orincrease. “Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or in oppositionto proliferative properties.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, the term “purify” means to make substantiallypure or clear from unwanted components, material defilement, admixtureor imperfection. “Purified” refers to the state of being pure.“Purification” refers to the process of making pure.

Region: As used herein, the term “region” refers to a zone or generalarea. In some embodiments, when referring to a protein or proteinmodule, a region may comprise a linear sequence of amino acids along theprotein or protein module or may comprise a three dimensional area, anepitope and/or a cluster of eptiopes. In some embodiments, regionscomprise terminal regions. As used herein, the term “terminal region”refers to regions located at the ends or termini of a given agent. Whenreferring to proteins, terminal regions may comprise N- and/orC-termini. N-termini refer to the end of a protein comprising an aminoacid with a free amino group. C-termini refer to the end of a proteincomprising an amino acid with a free carboxyl group. N- and/orC-terminal regions may there for comprise the N- and/or C-termini aswell as surrounding amino acids. In some embodiments, N- and/orC-terminal regions comprise from about 3 amino acid to about 30 aminoacids, from about 5 amino acids to about 40 amino acids, from about 10amino acids to about 50 amino acids, from about 20 amino acids to about100 amino acids and/or at least 100 amino acids. In some embodiments,N-terminal regions may comprise any length of amino acids that includesthe N-terminus, but does not include the C-terminus. In someembodiments, C-terminal regions may comprise any length of amino acids,that include the C-terminus, but do not comprise the N-terminus.

Region of antibody recognition: As used herein, the term “region ofantibody recognition” refers to one or more regions on one or moreantigens or between two or more antigens that are specificallyrecognized and bound by corresponding antibodies. In some embodiments,regions of antibody recognition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9or at least 10 amino acid residues. In some embodiments, regions ofantibody recognition comprise a junction between two proteins or betweentwo domains of the same protein that are in close proximity to oneanother.

Sample: As used herein, the term “sample” refers to an aliquot orportion taken from a source and/or provided for analysis or processing.In some embodiments, a sample is from a biological source such as atissue, cell or component part (e.g. a body fluid, including but notlimited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluidand semen). In some embodiments, a sample may be or comprise ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. In some embodiments, a sample is orcomprises a medium, such as a nutrient broth or gel, which may containcellular components, such as proteins or nucleic acid molecule. In someembodiments, a “primary” sample is an aliquot of the source. In someembodiments, a primary sample is subjected to one or more processing(e.g., separation, purification, etc.) steps to prepare a sample foranalysis or other use.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event. In someembodiments, a single unit dose is provided as a discrete dosage form(e.g., a tablet, capsule, patch, loaded syringe, vial, etc.).

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound or entity that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable. In some embodiments,stability is measured relative to an absolute value. In someembodiments, stability is measured relative to a reference compound orentity.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term typically means within about 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Target site: The term “target site” as used herein, refers to a regionor area targeted by a given compound, composition or method of theinvention. Target sites may include, but are not limited to cells,tissues, organs, organ systems, niches and the like.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition. In some embodiments, a therapeutically effectiveamount is provided in a single dose. In some embodiments, atherapeutically effective amount is administered in a dosage regimencomprising a plurality of doses. Those skilled in the art willappreciate that in some embodiments, a unit dosage form may beconsidered to comprise a therapeutically effective amount of aparticular agent or entity if it comprises an amount that is effectivewhen administered as part of such a dosage regimen.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in a 24 hr period. It may be administered as asingle unit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule or entity. Molecules or entities may undergo a series ofmodifications whereby each modified product may serve as the“unmodified” starting molecule or entity for a subsequent modification.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Protein Expression System

Protein expression is carried out using 293E cells. 293E cells areHEK293 cells stably expressing EBNA1 (Epstein-Barr virus nuclearantigen-1). These cells are human cells that post-translationally modifyproteins with human-like structures (e.g. glycans). Such cells areeasily transfectable and scalable and are able to grow to high densitiesin suspension culture. During protein production, 293E cells are grownin serum-free medium to facilitate down-stream purification. Some of theproteins produced comprise additional amino acids encoding one or moredetectable labels for purification [e.g. polyhistidine tag, flag tag(DYKDDDDK; SEQ ID NO: 265), etc.] Proteins are N-terminally labeled,C-terminally labeled and/or biotinylated.

Some of the proteins produced comprise additional amino acids encodingone or more 3C protease cleavage site (LEVLFQGP; SEQ ID NO: 266) Suchsites allow for cleavage between residues Q and G of the 3C proteasecleavage site upon treatment with 3C protease, including with rhinovirus3C protease. Cleavage sites are introduced to allow for removal ofdetectable labels from recombinant proteins.

Sequences encoding recombinant proteins of the present invention arecloned into pTT5 vectors (NRC Biotechnology Research Institute,Montreal, Québec.) for transfection into cells. Such vectors are small(˜4.4 kb), facilitate transient transfection, comprise a strong CMVpromoter for robust protein synthesis and comprise an oriP for episomalreplication in EBNA1-expressing cells.

Example 2. Generation of Antibodies

Antibodies Produced by Standard Monoclonal Antibody Generation

Antibodies are generated in knockout mice, lacking the gene that encodesfor desired target antigens. Such mice are not tolerized to targetantigens and therefore generate antibodies against such antigens thatmay cross react with human and mouse forms of the antigen. For theproduction of monoclonal antibodies, host mice are immunized withrecombinant proteins to elicit lymphocytes that specifically bind tothese proteins. Lymphocytes are collected and fused with immortalizedcell lines. The resulting hybridoma cells are cultured in a suitableculture medium with selection agents to support the growth of only fusedcells.

Desired hybridoma cell lines are then identified through bindingspecificity analysis of the secreted antibodies for the target peptideand clones of these cells are subcloned through limiting dilutionprocedures and grown by standard methods. Antibodies produced by thesecells are isolated and purified from the culture medium by standardimmunoglobulin purification procedures

Antibodies Produced Recombinantly

Recombinant antibodies are produced using the hybridoma cells producedabove. Heavy and light chain variable region cDNA sequences of theantibodies are determined using standard biochemical techniques. TotalRNA are extracted from antibody-producing hybridoma cells and convertedto cDNA by reverse transcriptase (RT) polymerase chain reaction (PCR).PCR amplification is carried out on the resulting cDNA using primersspecific for amplification of the heavy and light chain sequences. PCRproducts are then subcloned into plasmids for sequence analysis. Oncesequenced, antibody coding sequences are placed into expression vectors.For humanization, coding sequences for human heavy and light chainconstant domains are used to substitute for homologous murine sequences.The resulting constructs are transfected into mammalian cells capable oflarge scale translation.

Antibodies Produced by Using Antibody Fragment Display Library ScreeningTechniques

Antibodies of the present invention may be produced using highthroughput methods of discovery. Synthetic antibodies are designed byscreening target antigens using a phage display library. The phagedisplay libraries are composed of millions to billions of phageparticles, each expressing a unique Fab antibody fragment or singlechain variable fragment (scFv) on their viral coat. In Fab antibodyfragment libraries, the cDNA encoding each fragment contains the samesequence with the exception of a unique sequence encoding the variableloops of the complementarity determining regions (CDRs). The V_(H)chains of the CDR are expressed as a fusion protein, linked to theN-terminus of the viral pIII coat protein. The V_(L) chain is expressedseparately and assembles with the V_(H) chain in the periplasm prior toincorporation of the complex into the viral coat. Target antigens areincubated, in vitro, with members of phage display libraries and boundphage particles are precipitated. The cDNA encoding the CDRs of thebound Fab subunits is sequenced from the bound phage. The cDNA sequenceis directly incorporated into antibody sequences for recombinantantibody production, or mutated and utilized for further optimizationthrough in vitro affinity maturation.

Antibodies Produced Using Affinity Maturation Techniques

Fabs capable of binding target antigens are identified using thelibraries described above and high affinity mutants are derived fromthese through the process of affinity maturation. Affinity maturationtechnology is used to identify sequences encoding CDRs that have thehighest affinity for the target antigen. Using this technology, the CDRsequences isolated using the phage display library selection processdescribed above are mutated randomly as a whole or at specific residuesto create a millions to billions of variants. These variants areexpressed in Fab antibody fragment fusion proteins in a phage displaylibrary and screened for their ability to bind the target antigen.Several rounds of selection, mutation and expression are carried out toidentify antibody fragment sequences with the highest affinity for thetarget antigen. These sequences can be directly incorporated intoantibody sequences for recombinant antibody production.

Example 3. Identification and Characterization of Antibodies Directed toRecombinant Proteins

Recombinant proteins are synthesized according to the method of Example1 or obtained from commercial sources. Recombinant proteins expressedinclude those listed in Table 17.

TABLE 17 Recombinant proteins Recombinant Protein Key Features proTGF-β1C4S N-terminal association blocked TGF-β1 LAP C4S LAP only N-terminalassociation blocked proTGF-β1 complexed with N-terminal association withLTBP1 LTBP1S splice variant TGF-β1 LAP + sGARP LAP only N-terminalassociation with soluble GARP proTGF-β1 sGARP N-terminal associationwith soluble GARP proGDF-8 GDF-8 prodomain Prodomain only

Both human and non-human (including, but not limited to mouse) isoformsof the recombinant proteins listed in Table 17 are expressed.

Antibodies are generated according to the methods described in Example2, which bind to recombinant proteins expressed and are subjected toscreening to identify antibodies with desired binding properties. ELISAassays are used initially to identify antibody candidates thatdemonstrate affinity for desired antigens, while showing reduced or noaffinity for undesired antigens.

Identification of Stabilizing Antibodies Directed to the TGF-β1 GPC

Antibodies directed to proTGF-β1 C4S are screened using ELISAs to detectbinding to positive and negative selection antigens. Antibodies areassessed overall for their ability to associate with prodomains (with orwithout ligand) and decrease TGF-β signaling. ELISA plates are coatedwith neutravidin and incubated with biotinylated proTGF-β1 C4Srecombinant proteins. To identify and eliminate antibodies that bind tomiscellaneous elements (e.g. polyhistidine tags, flag tags and/or 3Cproteinase cleavage sites), coated ELISA plates are incubated with humanICAM-1 proteins comprising one or more of such miscellaneous elements.To identify and eliminate antibodies that bind to free TGF-β1 growthfactor and/or LAP, coated ELISA plates are incubated with human TGF-β1LAP C4S and/or TGF-β1 growth factor. Antibodies that may be specific formurine versions are identified by incubating coated ELISA plates withbiotinylated muproTGF-β1 C4S. Recombinant proteins that associate withantibodies bound on ELISA plates are detected using secondary antibodiesconjugated with enzymes for detection (e.g. colorimetric, fluorimetric)that bind to detectable labels present on bound recombinant proteins.Antibodies are selected for additional rounds of selection or eliminatedfrom testing pools based on results obtained.

Antibodies directed to proTGF-β1 C4S are further assessed for theirability to stabilize TGF-β1 GPCs. Cells expressing GPCs and/or αvβ₆integrin are incubated with selected antibodies and resultingsupernatants are used to treat cultures of cells comprisingTGF-β-responsive reporter constructs to detect free growthfactor-dependent gene expression activity. Additional assays are carriedout to characterize regions of antibody recognition bound by selectedantibodies as well as growth factor modulation in specific cell types(e.g. fibroblasts and/or T-cells). Finally, affinity binding estimatesare made using cross blocking experiments to bin antibodies as well asthrough the use of affinity analysis instruments, including, but notlimited to Octet® (ForteBio, Menlo Park, Calif.) family instruments.Antibodies are further selected based on their ability to stabilizealternative TGF-β GPC isoforms (e.g. TGF-β1, TGF-β2 and/or TGF-β3) andTGF-β1 GPCs from other species.

Identification of Releasing Antibodies Directed to Free TGF-β1 LAP

According to one mode for the generation of TGF-β1 GPC releasingantibodies, antibodies directed to proTGF-β1 LAP C4S are screened usingELISAs to detect binding to positive and negative selection antigens.Antibodies are assessed overall for their ability to associate with LAPand increase TGF-β1 free growth factor levels and/or signaling. ELISAplates are coated with neutravidin and incubated with biotinylatedproTGF-β1 LAP C4S recombinant proteins. To identify and eliminateantibodies that bind to miscellaneous elements (e.g. polyhistidine tags,flag tags and/or 3C proteinase cleavage sites), coated ELISA plates areincubated with human ICAM-1 proteins comprising one or more of suchmiscellaneous elements. To identify and eliminate antibodies that bindto free TGF-β1 growth factor, coated ELISA plates are incubated withhuman TGF-β1 growth factor. To identify and eliminate antibodies thatbind to latent TGF-β1, coated ELISA plates are incubated with humanTGF-β1 C4S. Antibodies that may be specific for murine versions areidentified by incubating coated ELISA plates with biotinylated muTGF-β1LAP C4S. Recombinant proteins that associate with antibodies bound onELISA plates are detected using secondary antibodies conjugated withenzymes for detection (e.g. colorimetric, fluorimetric) that bind todetectable labels present on bound recombinant proteins. Antibodies areselected for additional rounds of selection or eliminated from testingpools based on results obtained.

Antibodies directed to proTGF-β1 LAP C4S are further assessed for theirability to release TGF-β1 from GPCs. Cells expressing GPCs and/or αvβ₆integrin are incubated with selected antibodies and resultingsupernatants are used to treat cultures of cells comprisingTGF-β-responsive reporter constructs to detect free growthfactor-dependent gene expression activity. Additional assays are carriedout to characterize regions of antibody recognition bound by selectedantibodies as well as growth factor modulation in specific cell types(e.g. fibroblasts and/or T-cells). Finally, affinity binding estimatesare made using cross blocking experiments to bin antibodies as well asthrough the use of affinity analysis instruments, including, but notlimited to Octet® (ForteBio, Menlo Park, Calif.) family instruments.Antibodies are further selected based on their ability to elevate freegrowth factor relative to latent growth factor with alternative TGF-βGPC isoforms (e.g. TGF-β1, TGF-β2 and/or TGF-β3) and TGF-β1 GPCs fromother species.

Identification of Stabilizing Antibodies Directed to the TGF-β1 GPC inthe Context of LTBP

Antibodies directed to proTGF-β1 complexed with LTBP1 S are screenedusing ELISAs to detect binding to positive and negative selectionantigens. Antibodies are assessed overall for their ability to associatewith prodomains and decrease TGF-β signaling. ELISA plates are coatedwith neutravidin and incubated with biotinylated proTGF-β1 complexedwith LTBP1S antibody pools and incubated with recombinant proteinscomprising one or more detectable labels. To identify and eliminateantibodies that bind to miscellaneous elements (e.g. polyhistidine tags,flag tags and/or 3C proteinase cleavage sites), coated ELISA plates areincubated with human ICAM-1 proteins comprising one or more of suchmiscellaneous elements. To identify and eliminate antibodies that bindto free TGF-β1, coated ELISA plates are incubated with human TGF-β1growth factor. Antibodies that may be specific for murine versions areidentified by incubating coated ELISA plates with muproTGF-β1 complexedwith LTBP1S. Recombinant proteins that associate with antibodies boundon ELISA plates are detected using secondary antibodies conjugated withenzymes for detection (e.g. colorimetric, fluorimetric) that bind todetectable labels present on bound recombinant proteins. Antibodies areselected for additional rounds of selection or eliminated from testingpools based on results obtained.

Antibodies directed to proTGF-β1 complexed with LTBP1 S are furtherassessed for their ability to stabilize TGF-β1 GPCs against activationby αvβ6 expressed on cells. Cells expressing GPCs and/or αvβ6 integrinare incubated with selected antibodies and resulting supernatants areused to treat cultures of cells comprising TGF-β-responsive reporterconstructs to detect free growth factor-dependent gene expressionactivity. Additional assays are carried out to characterize regions ofantibody recognition bound by selected antibodies as well as growthfactor modulation in specific cell types (e.g. fibroblasts and/orT-cells). Finally, affinity binding estimates are made using crossblocking experiments to bin antibodies as well as through the use ofaffinity analysis instruments, including, but not limited to Octet®(ForteBio, Menlo Park, Calif.) family instruments. Antibodies arefurther selected based on their ability to stabilize alternative TGF-βGPC isoforms (e.g. TGF-β1, TGF-β2 and/or TGF-β3) and TGF-β1 GPCs fromother species.

Identification of Releasing Antibodies Directed to TGF-β1 LAP in theContext of GARP

Antibodies directed to TGF-β1 LAP complexed with sGARP are screenedusing ELISAs to detect binding to positive and negative selectionantigens. Antibodies are assessed overall for their ability to associatewith LAP, but not with free GARP and for their ability to increaseTGF-β1 free growth factor levels and/or signaling. ELISA plates arecoated neutravidin followed by incubation with biotinylated TGF-β1 LAPcomplexed with sGARP antibody pools and incubated with recombinantproteins comprising one or more detectable labels. To identify andeliminate antibodies that bind to miscellaneous elements (e.g.polyhistidine tags, flag tags and/or 3C proteinase cleavage sites),coated ELISA plates are incubated with human ICAM-1 proteins comprisingone or more of such miscellaneous elements. To identify and eliminateantibodies that bind to free GARP, coated ELISA plates are incubatedwith sGARP. Antibodies that may be specific for murine versions areidentified by incubating coated ELISA plates with muTGF-β1 LAP complexedwith sGARP. Recombinant proteins that associate with antibodies bound onELISA plates are detected using secondary antibodies conjugated withenzymes for detection (e.g. colorimetric, fluorimetric) that bind todetectable labels present on bound recombinant proteins. Antibodies areselected for additional rounds of selection or eliminated from testingpools based on results obtained.

Antibodies directed to TGF-β1 LAP complexed with sGARP are furtherassessed for their ability to release TGF-β1 from GPCs. Cells expressingGPCs and/or αvβ₆ integrin are incubated with selected antibodies andresulting supernatants are used to treat cultures of cells comprisingTGF-β-responsive reporter constructs to detect free growthfactor-dependent gene expression activity.

Antibodies are also tested for the ability to activate T-cell specificTGF-β-dependent gene expression. FoxP3 is a transcription factorexpressed in T-cells, known to be immunomodulatory. It is known to beregulated by TGF-β associated with T-cell surface GARP. Cells expressingGPCs as well as GARP are incubated with selected antibodies andresulting supernatants are used to treat cultures of EL4 cellscomprising FoxP3 reporter constructs.

Additional assays are carried out to characterize regions of antibodyrecognition bound by selected antibodies as well as growth factormodulation in specific cell types (e.g. fibroblasts and/or T-cells).Finally, affinity binding estimates are made using cross blockingexperiments to bin antibodies as well as through the use of affinityanalysis instruments, including, but not limited to Octet® (ForteBio,Menlo Park, Calif.) family instruments. Antibodies are further selectedbased on their ability to elevate free growth factor relative to latentgrowth factor with alternative TGF-β GPC isoforms (e.g. TGF-β1, TGF-β2and/or TGF-β3) and TGF-β1 GPCs from other species.

Identification of Stabilizing Antibodies Directed to the TGF-β1 GPC inthe Context of GARP

Antibodies directed to proTGF-β1 complexed with sGARP are screened usingELISAs to detect binding to positive and negative selection antigens.Antibodies are assessed overall for their ability to associate withprodomains and decrease TGF-β signaling. ELISA plates are coatedneutravidin, followed by incubation with biotinylated proTGF-β1complexed with sGARP antibody pools and incubated with recombinantproteins comprising one or more detectable labels. To identify andeliminate antibodies that bind to miscellaneous elements (e.g.polyhistidine tags, flag tags and/or 3C proteinase cleavage sites),coated ELISA plates are incubated with human ICAM-1 proteins comprisingone or more of such miscellaneous elements. To identify and eliminateantibodies that bind to free GARP, coated ELISA plates are incubatedwith human sGARP. Antibodies that may be specific for murine versionsare identified by incubating coated ELISA plates with muproTGF-β1complexed with sGARP. Recombinant proteins that associate withantibodies bound on ELISA plates are detected using secondary antibodiesconjugated with enzymes for colorimetric detection (e.g. horseradishperoxidase) that bind to detectable labels present on bound recombinantproteins. Antibodies are selected for additional rounds of selection oreliminated from testing pools based on results obtained.

Antibodies directed to proTGF-β1 complexed with sGARP are furtherassessed for their ability to stabilize TGF-β1 GPCs. Cells expressingGPCs are incubated with selected antibodies and resulting supernatantsare used to treat cultures of cells comprising TGF-β-responsive reporterconstructs to detect free growth factor-dependent gene expressionactivity.

Antibodies are also tested for the ability to reduce T-cell specificTGF-β-dependent gene expression. Cells expressing GPCs as well as GARPare incubated with selected antibodies and resulting supernatants areused to treat cultures of EL4 cells comprising FoxP3 reporterconstructs. Additional assays are carried out to characterize regions ofantibody recognition bound by selected antibodies as well as growthfactor modulation in specific cell types (e.g. fibroblasts and/orT-cells). Finally, affinity binding estimates are made using crossblocking experiments to bin antibodies as well as through the use ofaffinity analysis instruments, including, but not limited to Octet®(ForteBio, Menlo Park, Calif.) family instruments. Antibodies arefurther selected based on their ability to stabilize alternative TGF-βGPC isoforms (e.g. TGF-01, TGF-β2 and/or TGF-β3) and TGF-β1 GPCs fromother species.

Example 4. Chimeric Protein Design Using Sequence Alignments

For chimeric protein design, the alignment of TGF-β family members wasconstructed to identify conserved structural features and the degree ofconservation of these features (FIGS. 8A-8G.) Comparison betweenN-terminal region sequences revealed higher levels of conservation amongN-terminal regions of the prodomain. Based on this sequence alignmentand structural features of these protein modules, a generic chimericdesign strategy for TGF-β family members was adopted, such that chimeraswere designed where the ARM domains are swapped (either the entire ARMdomain, or subsets of the ARM domain as indicated) among family members.

Specifically, for the generation of chimeras comprising protein modulesof TGF-β1, TGF-β2 and/or TGF-β3, alignment of the three was carried outusing standard approaches, and these sequence alignments were used tocreate a homology model comparing TGF-β2 and TGF-β3 to the crystalstructure of porcine TGF-β1 (Shi, M. et al., Latent TGF-beta structureand activation. Nature. 2011 Jun. 15; 474(7351):343-9.) Briefly, thesequence of TGF-β2 or TGF-β3 was modeled based on the template structureand sequence alignment along with the satisfaction of standard spatialrestraints using standard procedures. These three dimensional modelswere analyzed to visualize how proposed chimeric combinations maycomprise areas of steric clash. As used herein, the term “steric clash”refers to an interaction between two or more entities and/or moietiesthat is disruptive to the shape and/or conformation of each entity, eachmoiety or an entity comprising the two or more moieties participating inthe interaction. Three dimensional modeling revealed possible stericclashes between the latency loop of TGF-β2 and the mature growth factorof TGF-β1. Specifically, the TGF-β2 latency loop comprises a D-Y-P aminoacid sequence, the side chains of which may overlap with regions of theTGF-β1 growth factor.

Example 5. TGF-β1 Chimeric Protein with TGF-β2 Trigger Loop

The activation mechanism for TGF-β2 remains to be fully understood.Activation may be dependent upon one or more associations between theTGF-β2 trigger loop and α₉β₁ integrin. To assess this mechanism ofTGF-β2 activity, chimeric proteins are synthesized comprising GPCscomprising TGF-β1 wherein protein modules comprising the sequenceSGRRGDLATI (SEQ ID NO:242) are substituted with protein modulescomprising TGF-β2 trigger loops comprising the sequenceGTSTYTSGDQKTIKSTRKK (SEQ ID NO:180) The activation mechanism of thesechimeric proteins (TGF-β1^(Trigger Loop (short)β2) chimeric proteins) istested by cell based assay. Cells (HEK293 or Sw-480 cells) aretransfected with or without α₉β₁ integrin in addition to either GPCscomprising TGF-β2, GPCs comprising TGF-β1^(Trigger Loop (short)β2)and/or GPCs comprising mutant TGF-β2 (as non-active controls) whereintrigger loops comprise the mutations Y240A, D245A and/or Q246A. Reportercell lines are used to detect growth factor release.

Example 6. Assessment of α9β1-TGF-β2 Binding and Growth Factor Release

Binding between α₉β₁ and TGF-β2 as well as subsequent growth factorrelease is not well understood in the art. If the residues involved inthis association can be elucidated, antibodies designed to disruptα₉β₁-TGF-β2 association may be developed and used to specifically targetTGF-β2 growth factor release.

Mutant constructs as well as chimeras comprising altered forms of TGF-β2are tested by activation assay so that the α₉β₁ binding site on TGF-β2may be mapped. This is done by generating TGF-β1/TGF-β2 chimeras withdeletion and/or mutation of amino acid residues in or around the triggerloop (in some embodiments, comprising the amino acid sequenceFAGIDGTSTYTSGDQKTIKSTRKKNSGKTP; SEQ ID NO: 65) or with residue-specificmutations to alanine. In some cases, TGF-β1 or TGF-β3 may or may notserve as negative controls for α₉β₁ binding. In some embodiments,recombinant proteins used for α₉β₁ binding site mapping may includethose listed in Table 18. These include proTGF-β2-M1, proTGF-β2-M2,proTGF-β2-M3, proTGF-β2-M4 and proTGF-β2-M5 comprising amino aciddeletions within the trigger loop. Also included is proTGF-β2-M6comprising mutation of two residues, Ile-Asp, to Phe-Thr. Finally, achimeric protein is included which comprises TGF-β2 wherein a portion ofthe trigger loop has been substituted with a portion of the trigger loopfrom TGF-β1.

TABLE 18 Recombinant protein for α9β1 binding site mapping SEQRecombinant ID Protein Key Features NO TGF-β2SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE   2VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKI EQLSNMIVKSCKCS proTGF-β2-M1SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 267VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS proTGF-β2-M2SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 268VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIV KSCKCS proTGF-β2-M3SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 269VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS proTGF-β2-M4SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 270VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQL SNMIVKSCKCS proTGF-β2-M5SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 271VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQL SNMIVKSCKCS proTGF-β2-M6SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 272VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGFTGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKI EQLSNMIVKSCKCSproTGF-β2^(RGDβ1) SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEE 273VPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDTGRRGDLATINSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIV KSCKCS

The activation mechanism of these recombinant proteins is tested by cellbased assay. Cells (HEK293 or Sw-480 cells) are transfected with orwithout α₉β₁ integrin in addition to either GPCs comprising TGF-β2, GPCscomprising alanine substitution mutations for each residue in thetrigger loop (wherein each GPC tested comprises a single substitution)one of the recombinant proteins listed in Table 18 and/or GPCscomprising inactive mutants of TGF-β2 (as non-active controls). Reportercell lines are used to detect growth factor release in media samplestaken from the transfected cells. Results are used to determine whichresidues within the trigger loop are necessary for α₉β₁-dependent TGF-β2growth factor release.

Example 7. Sequence Alignment

A multiple sequence alignment of TGF-β family members was adapted fromShi et. al. 2011 (Shi, M. et al., Latent TGF-beta structure andactivation. Nature. 2011 Jun. 15; 474(7351):343-9.) The sequences ofhuman TGF-β1, TGF-β2, TGF-β3, GDF-11, Inhibin Beta A, Inhibin Alpha A,BMP9, BMP2, BMP4, BMP7, BMP6, BMP8A, Lefty1, and murine TGF-β1, GDF11,GDF8 and cynomolgous monkey TGF-β1, and GDF8 were added to the alignmentusing standard methods, and the sequences included the full-lengthproteins (excluding signal peptide sequences) (FIGS. 8A-8G.)

Example 8. Myostatin Proliferation Assay

C₂C₁₂ murine myoblasts (ATCC, Manassas, Va.) are cultured in Dulbecco'smodified essential medium (DMEM; Life Technologies, Carlsbad, Calif.)with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, Calif.)prior to carrying out the assay. The percentage of FBS is varied and/orreplaced with bovine serum albumin (BSA) at varying concentrations. Cellproliferation assays are conducted in uncoated 96-well plates. C₂C₁₂cultures are seeded at 1000 cells per well. After allowing the cells toattach for 16 hours, myostatin test media is added. Recombinant humanmyostatin (R&D Systems, Minneapolis, Minn.) is used for standard curvegeneration. For experimental systems, the supernatant from 293E cellsoverexpressing myostatin is added, following treatment with experimentalantibodies. All samples are run in replicates of 8. Plates are incubatedfor 72 hours in an atmosphere of 37° C. and 5% CO₂. Proliferation isassessed using a CellTiter-Glo® Luminescent Cell Viability Assay(Promega BioSciences, LLC, Madison, Wis.) whereby cell lysis generates aluminescent signal proportional to the amount of ATP present, which isdirectly proportional to the number of cells present in culture (Thomas,M. et al., Myostatin, a negative regulator of muscle growth, functionsby inhibiting myoblast proliferation. 2000. 275(51):40235-43.)

Example 9. GPC Immobilization by Biotinylation and Detection ofIntegrin-Mediated Growth Factor Release

Recombinant GPCs of the present invention are N-terminally biotinylatedand incubated on streptavidin/avidin-coated culture surfaces. Cellsexpressing various integrins are added to the cell culture surfaces andcultured for 24 hours. Media are removed and added to growth factorreporter cell cultures that express luciferase in response to growthfactor activity. After 24 hours, cells are washed, lysed and analyzedfor luciferase activity.

Example 10. Protein Purification by Ni-NTA

Cells (293-6E cells) expressing His-tagged proteins are cultured inserum-free medium (FreeStyle F17 medium, Life Technologies, Carlsbad,Calif.) supplemented with 4 mM glutamine, 0.1% Pluronic F68 and 25 μg/mlG418. Once their viability drops below 50%, tissue culture supernatantis collected and cleared by centrifugation for 10 minutes at 200×gravityat 4° C. Supernatant is then filtered by passing it through a 0.22 or0.45 μm pore filter. Filtered supernatant is combined with Tris, NaCland NiCl₂ for a final concentration of 50 mM Tris pH 8.0, 500 mM NaCland 0.5 mM NiCl₂. 1 ml of the adjusted solution is collected for lateranalysis by SDS-poly acrylamide gel electrophoresis (PAGE) or Westernblot, while another portion of the adjusted solution is combined withwashed Ni-NTA resin (Life Technologies, Carlsbad, Calif.) at aconcentration of 5-10 ml of Ni-NTA resin per 300 ml of the adjustedsolution. This combined solution is then stirred at 4° C. using asuspended magnetic stir bar (to prevent grinding of Ni-NTA agarose.)Ni-NTA resin is next collected by centrifugation at 200×gravity at 4° C.for 10 minutes.

Next, the column is washed with 15 column volumes (CV) of wash buffer(20 mM Tris, pH 8.0, 500 mM NaCl and 20 mM imidazole.) An aliquot of thelast wash is collected for analysis. The column is then eluted with 3 CVof elution buffer (20 mM Tris, pH 8.0, 500 mM NaCl and 300 mM imidazole)and 1/3 column volume fractions are collected for analysis.

The absorbance at 280 nm is measured in each of the eluted fractionscollected and compared to the absorbance at 280 nm of blank elutionbuffer. Earlier fractions typically have negative absorption due to theimidazole gradient; however, fractions containing higher amounts ofprotein have positive values. Collected fractions are then run onSDS-PAGE for analysis and relevant fractions are pooled for furtherpurification.

Example 11. Design of GDF-8/GDF-11/Activin Chimeras

The structure-based alignment of TGF-β family members was used toconstruct three-dimensional models of potential chimeric proteinscomprising combinations of modules from GDF-8 and GDF-11 using theSchrodinger Bioluminate software. A chimeric model of GDF-8 comprisingan arm region of GDF-11 (SEQ ID NO:216) revealed a region of potentialsteric clash involving GDF-11 residue F95. According to the model, F95from the GDF-11 arm causes destabilization of the α2 helix of thechimeric GPC. Therefore, GDF8/GDF11/Activin chimeras were designed sothat the ARM region of the chimera contains the α2 helix.

Example 12. ELISA Analysis

Enzyme-linked immunosorbent assay (ELISA) analysis is carried out toassess antibody binding. 96-well ELISA assay plates are coated withneutravidin, a deglycosylated version of streptavidin with a moreneutral pI. Target proteins are expressed with or without histidine(His) tags and subjected to biotinylation. Biotinylated target proteinsare incubated with neutravidin-coated ELISA assay plates for two hoursat room temperature and unbound proteins are removed by washing threetimes with wash buffer (25 mM Tris, 150 mM NaCl, 0.1% BSA, 0.05%TWEEN®-20.) Primary antibodies being tested are added to each well andallowed to incubate at room temperature for 1 hour or more. Unboundantibody is then removed by washing three times with wash buffer.Secondary antibodies capable of binding to primary antibodies beingtested and conjugated with detectable labels are then incubated in eachwell for 30 minutes at room temperature. Unbound secondary antibodiesare removed by washing three times with wash buffer. Finally, boundsecondary antibodies are detected by enzymatic reaction, fluorescencedetection and/or luminescence detection, depending on the detectablelabel present on secondary antibodies being detected.

Example 13. Identification of Antibodies Using Phage Selection

Screening programs are conducted to generate antibody panels that bindtarget antigens. Antibody panel diversity is measured by epitopediversity as opposed to diversity of antibody sequences. Bothsolid-phase phage enrichment strategies as well as solution-phaseenrichment strategies are employed.

Target antigens (both for solid-phase and solution phase enrichment) aresubjected to biophysical characterization prior to use, includingreducing and non-reducing SDS-PAGE to establish purity and sizeexclusion chromatography (SEC) to establish acceptable aggregationlevels. Additionally, functional assays are carried out to verify targetantigen bioactivity.

2-3 rounds of enrichment are carried out with the expectation that onlythree rounds will be necessary. Aliquots of phage from selection rounds2-4 are preserved for later use. After enrichment, randomly selectedclones are screened by ELISA to examine binding to target antigens aswell as non-target antigens. Based on these analyses, up to 500 clonesare selected for nucleotide sequencing and analysis of the number ofdistinct antibodies as well as the frequency of isolation and number ofdistinct V_(H) and V_(L) regions. Based on these subsequent analyses, upto 100 clones are selected for epitope binning by epitope-relatednessusing surface plasmon resonance technology (or equivalent approach.)Dissociation constants (k_(off)) for each are determined and up to 50clones are selected for further characterization.

Final candidates are expressed as bivalent antibody constructs, purifiedand k_(off) for each are determined. Cell-based functional assays areused to characterize purified bivalent antibodies.

Example 14. Identification of Antibodies that Block Activation ofproGDF-8

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind proGDF-8 and block release of mature growth factor.Antibody generation is carried out according to the methods of Example13 wherein recombinant proGDF-8 is used for solid-phase enrichment andbiotinylated proGDF-8 is used for solution-phase enrichment. Antigenpreparations are tested for aggregation levels to ensure that >95% aredimeric species. In ELISA analysis of enriched clones, binding to sixantigens is assessed (proGDF-8, GDF-8 prodomain, GDF-8 growth factor,murine proGDF-8, proGDF-11 and proTGF-β1 C4S.) Clones selected based onELISA analysis are sequenced and antibodies are developed according tothe methods of Example 13.

Example 15. Identification of Antibodies that Activate the Release ofGDF-11 Growth Factor from the Latent GPC

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind the prodomain of GDF-11 and activate the release ofmature growth factor. Antibody generation is carried out according tothe methods of Example 13 wherein recombinant GDF-11 prodomain is usedfor solid-phase enrichment and biotinylated GDF-11 prodomain is used forsolution-phase enrichment. Antigen preparations are tested foraggregation levels to ensure that >95% are monomeric species. In ELISAanalysis of enriched clones, binding to six antigens is assessed (GDF-11prodomain, proGDF-11, GDF-11 growth factor, GDF-8 prodomain, murineGDF-11 prodomain and proTGF-β1 C4S.) Clones selected based on ELISAanalysis are sequenced and antibodies are developed according to themethods of Example 13.

Example 16. Identification of Antibodies that Activate the Release ofTGF-β1 from the proTGF-β1/GARP Complex

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind TGF-β1 LAP that is complexed with sGARP (TGF-β1LAP-sGARP) and activate the release of mature growth factor. Antibodygeneration is carried out according to the methods of Example 13 whereinrecombinant biotinylated TGF-β1 LAP-sGARP is used for solid-phaseenrichment and biotinylated TGF-β1 LAP-sGARP is used for solution-phaseenrichment. Antigen preparations are tested for aggregation levels toensure that >95% of the species comprise dimeric TGF-β1 LAP complexedwith monomeric sGARP. In ELISA analysis of enriched clones, binding toeight antigens is assessed (TGF-β1 LAP-sGARP, proTGFb1-sGARP, sGARP,TGF-β1 LAP C4S, proTGF-β1 C4S, LTBP1-proTGFb1, ICAM-1 N-His, ICAM-1C-His.) Clones selected based on ELISA analysis are sequenced andantibodies are developed according to the methods of Example 13.

Example 17. Identification of Antibodies that Block the Release ofMature Growth Factor from the proTGF-β1/GARP Complex

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind to the complex formed by proTGF-β1 and GARP(proTGF-β1-GARP) and inhibit release of mature growth factor. Antibodygeneration is carried out according to the methods of Example 13 whereinrecombinant biotinylated proTGF-β1-sGARP is used for solid-phaseenrichment and biotinylated proTGF-β1-GARP is used for solution-phaseenrichment. Antigen preparations are tested for aggregation levels toensure that >95% of the species comprise dimeric proTGF-β1 complexedwith monomeric sGARP. In ELISA analysis of enriched clones, binding toeight antigens is assessed (proTGF-β1-GARP, TGF-β1 LAP, proTGF-β1 C4S,proTGF-β1/LTBP1S complex, TGF-β1 LAP-sGARP, sGARP, ICAM-1 C-His, ICAM-1N-His.) Clones selected based on ELISA analysis are sequenced andantibodies are developed according to the methods of Example 13.

Example 18. Identification of Antibodies that Block the Release ofTGF-β1 from proTGF-β1 Complexed with LTBP1S

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind proTGF-β1 complexed with LTBP1S (proTGF-β1-LTBP1S)and inhibit release of mature growth factor. Antibody generation iscarried out according to the methods of Example 13 wherein recombinantproTGF-β1-LTBP1S is used for solid-phase enrichment and biotinylatedproTGF-β1-LTBP1S is used for solution-phase enrichment. Antigenpreparations are tested for aggregation levels to ensure that >95% ofthe species comprise dimeric proTGF-β1 complexed with monomeric LTBP1S.In ELISA analysis of enriched clones, binding to eight antigens isassessed (proTGF-β1-LTBP1S, TGF-β1 LAP, TGF-β1 growth factor, proTGF-β1C4S, murine proTGF-β1-LTBP1S, LTBP1S, GDF-8 prodomain and proTGF-β2.)Clones selected based on ELISA analysis are sequenced and antibodies aredeveloped according to the methods of Example 13.

Example 19. Identification of Pan-Specific Antibodies that Block theRelease of TGF-β1 from proTGF-β1

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind proTGF-β1 and inhibit the release of mature growthfactor. Antibody generation is carried out according to the methods ofExample 13 wherein recombinant proTGF-β1 is used for solid-phaseenrichment and biotinylated proTGF-β1 is used for solution-phaseenrichment. Antigen preparations are tested for aggregation levels toensure that >95% are dimeric species. In ELISA analysis of enrichedclones, binding to seven antigens is assessed (TGF-β1 LAP, TGF-β1 growthfactor, proTGF-β1 C4S, murine proTGF-β1 C4S, GDF-8 prodomain andproTGF-β2.) Clones selected based on ELISA analysis are sequenced andantibodies are developed according to the methods of Example 13.

Example 20. Identification of Pan-Specific Antibodies that Activate theRelease of TGF-β1 from proTGF-β1

Production of a diverse panel of antibodies is carried out to identifyantibodies that bind TGF-β1 LAP and activate the release of maturegrowth factor. Antibody generation is carried out according to themethods of Example 13 wherein recombinant TGF-β1 LAP C4S is used forsolid-phase enrichment and biotinylated TGF-β1 LAP C4S is used forsolution-phase enrichment. Antigen preparations are tested foraggregation levels to ensure that >95% are dimeric species. In ELISAanalysis of enriched clones, binding to seven antigens is assessed(TGF-β1 LAP C4S, proTGF-β1 C4S, murine proTGF-β1 C4S, TGF-β1 maturegrowth factor, proGDF-8 and proTGF-β2.) Clones selected based on ELISAanalysis are sequenced and antibodies are developed according to themethods of Example 13.

Example 21. Immunization of TGF-β1 Knockout Mice

Neonatal mice are immunized according to the methods of Oida et al(Oida, T. et al., TGF-β induces surface LAP expression on Murine CD4 Tcells independent of FoxP3 induction. PLOS One. 2010. 5(11):e15523, thecontents of which are herein incorporated by reference in theirentirety.) TGF-β-deficient neonatal mice receive galectin-1 injectionsto prolong survival (typically 3-4 weeks after birth in these mice.)Cells stabily producing antigenic proteins (e.g. proTGF-β1-GARP orTGF-β1 LAP-GARP; 1-4×106 cells in 10-25 μl PBS) or purified antigenicproteins are used to immunize the mice every other day byintraperitoneal injection for 10 days beginning on the 8^(th) day afterbirth. Spleen cells are harvested on day 22 after birth. Harvestedspleen cells are fused with SP 2/0 myeloma cells. Resulting hybridomacells are assessed for successful production of anti-proTGF-β1antibodies.

Example 22. Expression of TGF-β1 Complexes and Protein Analysis

proTGF-β1 expression was carried out with or without His-tagged LTBP1Sor sGARP according to the methods of Example 10. proTGF-β1 expressedwithout LTBP1S or sGARP comprised C4S mutation to prevent prodomainassociation with these factors and an N-terminal His tag. Purifiedproteins were analyzed by SDS-PAGE under either reducing or non-reducingconditions (to maintain protein dimers or complexes). FIG. 11 depictsthe results indicating successful expression of these proteins andprotein complexes.

Example 23. Cell-Based Antigen Expression of TGF-β1/GARP Complexes

Pro B-cell lymphoma cell lines were developed that stably express both(membrane-bound) GARP and proTGF-β1 or TGF-β1 LAP. Membrane-associatedGARP was cloned into pYD7 vector (NRC Canada, Ottawa, CA) whileproTGF-β1 and TGF-β1 LAP were cloned into pcDNA3.1 vectors (LifeTechnologies, Carlsbad, Calif.) These vectors allow for blasticidin andG418-based selection, respectively. Pre-B-cell lymphoma-derived cellsfrom BALB/c swiss mice (referred to herein as 300.19 cells) weretransfected with empty vector control or GARP with coexpression ofeither proTGF-β1 or TGF-β1 LAP and selected with G418 plus blasticidin.Resistant cells were subcloned and single colonies were selected. Cellscultured from resulting cell lines were probed with antibodies(conjugated with fluorescent particles) directed to expressed proteinsand examined by flow cytometry for fluorescence intensity. FIG. 12displays fluorescence intensity data collected from resulting cells.Baseline values associated with cells transfected with empty vectorcontrol are shown in FIG. 12A, while elevated fluorescence intensity inFIGS. 12B and 12C indicate cell surface expression of GARP complexes.Quantification of surface-expressed proteins was carried out throughadditional analyses in which the same fluorescently labeled cells usedto generate the data depicted in FIG. 12, were examined by flowcytometry alongside beads with defined antibody binding capacity for thegeneration of a standard curve. These beads were labeled with the sameantibodies used for labeling cells and fluorescence values obtained wereused to extrapolate the number of antibodies bound to surface expressedproteins. 300.19 cells expressing proTGF-β1-GARP were determined toexpress about 83,000 copies/cell, while 300.19 cells expressing TGF-131LAP-GARP were determined to express about 66,000 copies/cell.

Cell lines were next tested for TGF-β1 activity in the presence of cellsexpressing αvβ6 integrins, known to release TGF-β1 growth factor fromlatent GPCs. Conditioned media from these co-cultures was used to treatreporter cells comprising TGF-β receptors as well as the luciferasegene, driven by a TGF-β-responsive promoter, PAI-1. This was done in thepresence or absence of a neutralizing antibody, anti-TGF-β, clone 1D11.Resulting luciferase activity was assessed by luminometry. Resultsindicate that conditioned media from cells expressing empty vectors andTGF-β1 LAP-GARP complexes were unable to induce luciferase expressionwhen compared to baseline values, while conditioned media from cellsexpressing proTGF-β1-GARP displayed an enhanced ability to induceluciferase expression (see FIG. 12D.)

Example 24. Cell-Based Antigen Expression of proTGF-β1-LTBP1

NIH 3T3 mouse fibroblasts are developed that stably expressproTGF-β1-LTBP1. These secreted proteins bind to the cell surface or aredeposited in the extracellular matrix.

Example 25. LTBP3 Expression

Recombinant LTBP3 proteins are expressed with or without variousmodules, fragments, N-terminal secretion signal sequences (e.g. SEQ IDNO: 257) and/or N- or C-terminal histidine tags. Modules included insome expressed proteins include those listed in Table 19.

TABLE 19 LTBP3 modules SEQ ID Protein Sequence NO LTBP3 DIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLL 274 EGF- ECVDVDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQ like RQCLSP domain, module 1LTBP3  DVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDC 275 EGF-  QLP likedomain, module 2 LTBP3  DIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQH 276EGF-  GCE like domain, module 3 LTBP3 DIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLL 277 EGF-  ECV like domain,module 4 TB   KKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHC 278 domain,EIYPCPVYSSAEFHSLCP module 1 TB   DVCWSQRGEDGMCAGPLAGPALTFDDCCCRQGRGWGAQ279 domain, CRPCPPRGAGSHCP module 2

LTBP3 fragments included in some expressed proteins include those listedin Table 20.

TABLE 20 LTBP3 fragments SEQ Pro- ID tein Sequence NO L3- KKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHC 280 TB3TB4EIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDID iso- ECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVD form 1VDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDCCCRQGRGWGAQCRPCPPRGAGSHCPTSQSE L3- KKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWGDHC 281 TB3TB4EIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDID iso- ECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVD form 2VDECLDESNCRNGVCENTRGGYRCACTPPAEYSPAQRQCLSPEEMERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDC CCRQGRGWGAQCRPCPPRGAGSHCPTSQSEL3-  DIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQH 282 ETB3E,GCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCS type 1LGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFY YDGNLLECVDVDE L3- QDIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQ 283 ETB3E,HGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCC type 2SLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGF YYDGNLLECVDVDE L3- DIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQH 284 ETB3E,GCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCCS type 3LGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFY YDGNLLECV L3- QDIDECSQDPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQ 285 ETB3E,HGCEEVEQPHHKKECYLNFDDTVFCDSVLATNVTQQECCC type 4SLGAGWGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGF YYDGNLLECV

Further proteins expressed include those listed in Table 21.

TABLE 21 LTBP3 recombinant proteins SEQ Pro- ID tein Sequence NO L3- MDMRVPAQLLGLLLLWFSGVLGKKECYLNFDDTVFCDSV 286 TB3TB4LATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLC iso-PDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKC form VNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNG 1VCENTRGGYRCACTPPAEYSPAQRQCLSPEEMDVDECQDPAACRPGRCVNLPGSYRCECRPPWVPGPSGRDCQLPESPAERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDCCCRQ GRGWGAQCRPCPPRGAGSHCPTSQSEHHHHHHL3-  MDMRVPAQLLGLLLLWFSGVLGKKECYLNFDDTVFCDSV 287 TB3TB4LATNVTQQECCCSLGAGWGDHCEIYPCPVYSSAEFHSLC iso-PDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKC form VNTQPGYECYCKQGFYYDGNLLECVDVDECLDESNCRNG 2VCENTRGGYRCACTPPAEYSPAQRQCLSPEEMERAPERRDVCWSQRGEDGMCAGPLAGPALTFDDCCCRQGRGWGAQC RPCPPRGAGSHCPTSQSEHHHHHH L3- MDMRVPAQLLGLLLLWFSGVLGDIDECSQDPSLCLPHGA 288 ETB3E,CKNLQGSYVCVCDEGFTPTQDQHGCEEVEQPHHKKECYL type NFDDTVFCDSVLATNVTQQECCCSLGAGWGDHCEIYPCP 1CVYSSAEFHSLCPDGKGYTQDNNIVNYGIPAHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDGNLLECVDVD EHHHHHH His- MDMRVPAQLLGLLLLWFSGVLGHHHHHHSSGDIDECSQD 289 L3-PSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQ ETB3E,PHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWG typeDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPA 1NHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDG NLLECVDVDE His- MDMRVPAQLLGLLLLWFSGVLGHHHHHHSSGQDIDECSQ 290 L3-DPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVE ETB3E, QPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGW typeGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIP 2AHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYD GNLLECVDVDE His- MDMRVPAQLLGLLLLWFSGVLGHHHHHHSSGDIDECSQD 291 L3-PSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVEQ ETB3E, PHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGWG typeDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIPA 3HRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYDG NLLECV His- MDMRVPAQLLGLLLLWFSGVLGHHHHHHSSGQDIDECSQ 292 L3-DPSLCLPHGACKNLQGSYVCVCDEGFTPTQDQHGCEEVE ETB3E, QPHHKKECYLNFDDTVFCDSVLATNVTQQECCCSLGAGW typeGDHCEIYPCPVYSSAEFHSLCPDGKGYTQDNNIVNYGIP 4AHRDIDECMLFGSEICKEGKCVNTQPGYECYCKQGFYYD GNLLECV

Example 26. 293T CAGA-Luciferase Assay for GDF-8 Activity

CAGA-luciferase assays are carried out to test antibodies that modulateGDF-8 activity. A 50 μg/ml solution of fibronectin is prepared and 100μl are added to each well of a 96-well plate. Plates are incubated for30 min at room temperature before free fibronectin is washed away usingPBS. 293T cells comprising transient or stable expression of pGL4(Promega, Madison, Wis.) under the control of a control promoter orpromoter comprising smadl/2 responsive CAGA sequences are then used toseed fibronectin-coated wells (2×10⁴ cell/well in complete growthmedium.) The next day, cells are washed with 150 of cell culture mediumwith 0.1% bovine serum albumin (BSA) before treatment with GDF-8 with orwithout test antibody. Cells are incubated at 37° for 6 hours beforedetection of luciferase expression using BRIGHT-GLO™ reagent (Promega,Madison, Wis.) according to manufacturer's instructions.

Example 27. Detection of Myogenin Expression by FACS

257384 Lonza cells (Lonza, Basel, Switzerland) are plated in 24-wellplates at 4×10⁴ cells/well. The next day, cell media is replaced withdifferentiation media [dulbecco's modified eagle medium (DMEM)/F12 with2% horse serum.] Varying concentrations of GDF-8 are also included indifferentiation media in the presence or absence of test antibodies.Cells are then allowed to differentiate for 3 days.

After the 3 day period, differentiation status of each well is analyzedthrough analysis of myogenin expression levels. Cells from eachtreatment group are pooled and subjected to treatment using theTranscription Factor Buffer Set from BD Pharmingen (BD Biosciences,Franklin Lakes, N.J.), product number 562574 according to manufacturersinstructions. After fixation and permeabilization, 5 μl of phycoerythrin(PE)-myogenin or 1.25 μl of PE-control are added to the cells andincubated at 4° C. for 50 mins. Cells are then washed and resuspended inFACS buffer before analysis of cellular fluorescence by FACS.

Example 28. HT2 Cell Proliferation Assay

Antibodies are tested for the ability to modulate TGF-β activity usingan HT2 cell proliferation assay. HT2 cell proliferation inIL-4-containing medium is reduced in the presence of free TGF-β growthfactor. Antibodies with the ability to modulate free growth factorlevels by stabilizing TGF-β GPCs or by promoting the release and/oraccumulation of free growth factor may be tested using the HT2 culturesystem described here. Cells expressing proTGF-β are co-cultured withcells expressing αvβ₆ integrins. Cultures are treated with variousconcentrations of test antibody, purified TGF-β1 (as a positive control)or anti-TGF-β antibody 1D11 (R&D Systems, Minneapolis, Minn.) as anegative control.

HT2 cells are cultured in growth media (RPMI 1640, 10% FBS, 1% P/S, 4 mMGln, 50 μM beta-mercaptoethanol and 10 ng/mL IL-2) at 1.5×10⁵ cells/mlto ensure that cells are in log growth phase on the following day. Thenext day, cell supernatants being tested are diluted in HT2 assay media(RPMI 1640, 10% FBS, 1% P/S, 4 mM Gln, 50 μM beta-mercaptoethanol and7.5 ng/mL IL-4.) Growth media is removed from HT2 cell cultures andcells are washed with cytokine free media. Diluted supernatants areadded to each HT2 cell culture well and HT2 cells are cultured for 48hours at 37° C. and 5% CO₂. Cell viability in the HT2 cell cultures isthen determined using CELL-TITER GLO® reagent (Promega, Madison, Wis.)according to manufacturers instructions. Results are obtained asrelative light units (RLUs) which correlate with cell viability.

Example 29. Analysis of Recombinantly Expressed GDF-8

Histidine-tagged proGDF-8 was expressed according to the methods ofExample 10. Purified proteins were analyzed by SDS-PAGE under eitherreducing or non-reducing conditions (to maintain protein dimers). FIG.13 depicts the results indicating successful expression of theseproteins and protein complexes.

Example 30. TGF-β2 Chimeras

Chimeric proteins are synthesized that comprise TGF-β2 with arm regionsubstitutions from TGF-β1 and TGF-β3. The chimeric proteins alsocomprise N-terminal C5S mutations. These expressed chimeric proteins(listed in Table 22) have improved stability over some other chimericproteins.

TABLE 22 TGF-β2 chimeric proteins. Pro- Pro- tein tein SEQ mod- mod- IDule 1 ule 2 Chimeric Sequence NO TGF- TGF-SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 293 β2 β1DYPEPEEVPPEVLALYNSTRDRVAGESAEPEPEPE LAP armADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMF re-FNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVE gionLYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGV VRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHL QSSRHRR TGF- TGF-SLSTSSTLDMDQFMRKRIEAIRGQILSKLKLTSPPE 294 β2 β3DYPEPEEVPPEVLALYNSTRELLEEMHGEREEGCT LAP armQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGI re-TSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSK gionRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAE WLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRL KKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKR

The invention claimed is:
 1. An antibody produced by a method comprisingthe steps of: i. expressing mammalian proGDF-8; ii. subjecting theexpressed mammalian proGDF-8 to enzymatic cleavage with one or more offurin, bone morphogenetic protein-1 (BMP-1), mammalian tolloid protein(mTLD), mammalian tolloid-like 1 (mTLL1), and mammalian tolloid-like 2(mTLL2), so as to form a growth factor prodomain complex (GPC); iii.carrying out solid-phase or solution-phase enrichment with an antibodyfragment phage display library, wherein the GPC formed by enzymaticcleavage is used as a target antigen; iv. selecting phage particles thatbind to the GPC formed by enzymatic cleavage and excluding phageparticles that bind to isolated GDF-8 growth factor or isolated GDF-8prodomain; and v. producing recombinant antibodies havingcomplementarity determining regions (CDRs) of an antibody fragmentexpressed at the surface of the selected phage particles.
 2. Theantibody of claim 1, wherein said antibody is human or humanized.
 3. Theantibody of claim 1, wherein said antibody is subjected to affinitymaturation.
 4. The antibody of claim 1, wherein said method furthercomprises: vi. screening the recombinant antibodies and selecting thosewhich inhibit release of GDF-8 growth factor from GDF-8 GPCs.
 5. Theantibody of claim 4, wherein antibodies that inhibit GDF-8 activity areselected.
 6. The antibody of claim 1, wherein said method furthercomprises: vi. conducting a negative selection to remove antibodies thatbind to one or more undesired antigens.
 7. The antibody of claim 6,wherein said one or more undesired antigens are selected from the groupconsisting of GDF-8 prodomain, GDF-8 growth factor, murine proGDF-8,proGDF-11, proTGF-β1, and a protein with an amino acid sequence selectedfrom the group consisting of SEQ ID Nos: 207-230.
 8. The antibody ofclaim 7, wherein said antibody is human or humanized.
 9. The antibody ofclaim 7, wherein said antibody is subjected to affinity maturation. 10.The antibody of claim 1, wherein phage particle binding is determinedusing one or more binding assays selected from the group consisting ofenzyme-linked immunosorbent assays, surface plasmon resonance assays,and flow cytometry assays.
 11. The antibody of claim 1, wherein saidenzymatic cleavage comprises sequential enzymatic cleavage with at leasttwo enzymes.
 12. The antibody of claim 11, wherein said antibody ishuman or humanized.
 13. The antibody of claim 11, wherein said antibodyis subjected to affinity maturation.
 14. The antibody of claim 1comprising an isotype selected from the group consisting of IgG1, IgG2,IgG3, IgG4, IgA, IgGA2, IgD, IgE, and IgM.
 15. The antibody of claim 1comprising a bispecific antibody.
 16. The antibody of claim 1, whereinsaid mammalian proGDF-8 comprises the amino acid sequence of SEQ ID NO:5.